Chapter 16 Using Biotinylated Proteins to Demonstrate Immunodetection of Antigens via Western Blotting, Dot Blots, and Immunohistochemistry Thomas Millar, Ronald Knighton, and Jo-Anne Chuck Abstract Using biotinylated targets for detection by enzyme-linked avidin allows immunodetection methods to become more economic in cost and time as it negates the need for a specific primary antibody. Methods are described to use exogenously added biotin to complex biological samples to demonstrate western blotting, dot blots, and immunohistochemistry. These methods can be used in biological science tertiary teaching laboratories to allow novices to gain skills in a risk-free environment to promote student motivation and engagement. Key words Immunodetection, Undergraduate practical class, Biotinylation, Western blot, Immunochemistry, Dot blots, Inquiry-based learning, Electrophoresis
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Introduction The immunodetection methods outlined in this chapter exploit the affinity between the small molecule biotin (vitamin B7) and the protein avidin. Biotin is a naturally occurring molecule typically made by the microbiota in the human gut and is an important co-factor in many biochemical reactions. However biotin is used extensively in biotechnology in immunodetection processes [1]. Exploiting the very high affinity of the molecule to avidin, biotin-avidin-enzyme complexes can be detected using substrates that produce colorimetric products. This reporting system is commonly used in blots, immunohistochemistry, and ELISAs. In this study, the molecule is exogenously added to complex biological samples for detection to demonstrate these modern immunological methods. As the selectivity and detection of molecule–ligand interactions is used in a range of different biology laboratories, it is a very important technique to demonstrate to undergraduate science students [2, 3]. Beyond its use in diagnostics, the technique is
Biji T. Kurien and R. Hal Scofield (eds.), Detection of Blotted Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 1314, DOI 10.1007/978-1-4939-2718-0_16, © Springer Science+Business Media New York 2015
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often central to many protein chemistry, histology, and cell biology research projects. Both medical and biological science undergraduate students need to develop skills for carrying out these common methodologies. However, for higher education institutions, running practical classes teaching these skills is almost impossible because of the financial burden, time constraints, resource limitations, and obtaining a realistic outcome within the hands of novices [2, 3]. To accommodate students handling small volumes of very expensive reagents in experiments that take longer than a typical 4 h undergraduate laboratory, sessions often become mainly demonstrations, with key steps illustrated by prepared samples. Using the detection of biotin to illustrate this process, crucial steps such as incubations, blocking, and washing with shorter time periods are possible without the risk of interfering with the quality of the results obtained. Having a failsafe technique where the outcome is assured means that the educational process could be freed up to transfer ownership of the practical to the students [4].
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Materials Prepare all reagents using deionized water.
2.1 Gel Electrophoresis
1. Gel electrophoresis apparatus: There are many available with slightly different formats, e.g., Mini-Protean III (BioRad Herclues, CA, USA). 2. Other specialist equipment: Microcentrifuge, shaking or rocking platform, magnetic stirrer, boiling water bath, power packs, micro-pipettes, microcentrifuge tubes and racks (including those that float in water baths). 3. Resolving gel buffer: 1.5 M Tris–HCl (pH 8.8): Weigh out 18.2 g Tris base and dissolve in 80 mL water using a stirrer. Adjust to pH 8.8 with concentrated HCl and make up to 100 mL with water. Store at 4 °C (need approx. 3 mL per gel). 4. Stacking gel buffer: 0.5 M Tris–HCl (pH 6.8): Weigh 6 g Tris base and dissolve in 60 mL water using a stirrer. Adjust to pH 6.8 with concentrated HCl and make up to 100 mL with water. Store at 4 °C (need approx. 3 mL per gel). 5. 10 % (w/v) SDS: Using a balance in fume hood, weigh out 10 g of SDS. Make up to 100 mL using water. Store at room temperature (need 200 µL per gel). 6. 30 % acrylamide/Bis solution (29:1 acrylamide: Bis, 3.3 % cross-linker concentration): purchase premixed (BioRad Hercules, CA USA), store at 4 °C (need approx. 4 mL per gel) (see Note 1). 7. 10 % (w/v) ammonium persulfate: Weigh out 1 g and make up to 10 mL with water. Store at room temperature (need 100 µL per gel) (see Note 2).
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8. N,N,N′,N′-tetramethyl-ethylenediamine (TEMED): Store at 4 °C (see Note 3) (need 30 µL per gel). 9. SDS-PAGE running buffer: 0.025 M Tris–HCl, pH 8.3, 0.192 M glycine, 0.1 % SDS. To make a 10× stock, weigh out 30 g of Tris base, 144 g of glycine, 1 g of SDS and make up to 1 L with water (pH should not have to be adjusted). Dilute one part in ten in water before use. 10. Sample buffer: Add water (3.55 mL), 0.5 M Tris–HCl, pH 6.8 (1.25 mL), glycerol (2.5 mL), 10 % SDS (2.0 mL), and bromophenol blue (0.2 mL of a 0.5 % (w/v) solution in water). Store at room temperature. Add 50 µL of mercaptoethanol to 950 µL of sample buffer just before use (see Note 4). 11. Coomassie blue stain: 0.1 % (w/v) Coomassie blue in 40 % (v/v) methanol, 10 % (v/v) acetic acid, and 50 % (v/v) water. 12. Destaining solution: Add concentrated acetic acid (250 mL), methanol (1 L), and water (1,250 mL). 13. Protein molecular mass markers: Many are available including Precision Plus Protein Kaleidoscope Standards 10–250 kDa, SDS-PAGE Broad range Standards (unstained), (6.5–200 kDa). 14. 70 % (v/v) ethanol. 15. Samples: (a) Bovine serum (or horse serum) with 350 µg/mL biotinylated bovine serum albumin (B-BSA) (see Note 5). Weigh out 2.5 mg of B-BSA and make up to 250 µL with water. Add 35 µL to 1 mL of serum. (b) Bovine serum albumin (BSA, 0.5 mg/mL): Weigh out 5 mg of BSA and dissolve in 10 mL of water. (c) B-BSA (350 µg/mL). Take 35 µL of the 10 mg/mL solution of B-BSA and make up to 1 mL with water. (d) Negative control serum: use bovine serum (or horse serum). 2.2 Western Blotting and Immunoblots
1. Wet protein transfer apparatus: Many are available, e.g., Mini Trans-Blot (BioRad Hercules, CA USA). These will include gauze pads as support for the gel transfer assembly. Fill the cooling units with water and store at −20 °C. 2. PVDF membrane (one per gel). Cut to the size of gel (see Note 6). 3. Filter paper (two per gel) cut to the same size as gauze pads. 4. Transfer buffer: 25 mM Tris, pH 8.3, 192 mM glycine, 20 % (v/v) methanol, and 0.5 % SDS. Weigh out, Tris Base 3 g/L, glycine 14.4 g/L, methanol 200 mL/L, SDS 5 g/L and make up to 1 L with water. Do not adjust pH. Store and use at 5 °C. Approx. 1 L of buffer per tank is required.
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5. Tris Buffered Saline (TBS): 50 mM Tris–HCl pH 7.4, 150 mM NaCl (see Note 7). Typically this is made up as a 5× stock which is diluted before use. Weigh out 30.2 g Tris and 43.5 g of NaCl and dissolve in 800 mL of water. Adjust pH to 7.4 using concentrated HCl. Make up to 1 L. Before use, dilute one part in four using water. 6. Blocking solution: 5 % (w/v) skim milk power in TBS (see Note 7). Store at 4 °C. 7. Square plastic weigh boats approx. 15 cm × 2.5 cm and a shallow tray approx. 40 × 20 × 10 cm (see Note 8). 8. Glass rod (20 cm × 0.5 cm). 9. SIGMA-FAST BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium) tablets. 10. ExtrAvidin-alkaline phosphatase. 2.3 Immunohistochemistry
1. One mature rat. Strain is not important. 2. S-(+)-Ketamine hydrochloride. 3. Xylazine hydrochloride. 4. Ethanol. 5. Xylenes. 6. Paraplast wax for histology. 7. Sterile water (Note: Since this procedure is nonrecovery anesthesia, boiled water would suffice). 8. 5,000 IU/mL heparin. 9. Saline: Make up 1 L of saline by adding 9 g of NaCl to 1 L of sterile water and mixing. 10. Perfusion set and stand: This is a bottle (1 L) that can be hung upside down on a stand 1 m above the animal to be perfused. A hole is made in the lid of the bottle to allow a 5 mm diameter cannula to be poked through the lid so that it does not leak. A ~2 m tube is attached to the free end of cannula and a 16 gauge needle is attached to this for inserting into the left ventricle of the rat heart. 11. Biotin (100 mg). 12. Syringes: 1 and 50 mL with 20 gauge needles. 13. 5 % serum: Take 5 mL of bovine serum and dilute it with 95 mL of saline solution. 14. Primary antibody stock solution: 0.5 mL of bovine serum with 9.5 mL of saline solution. About 1 mL is put into a vial and labeled “stock primary antibody solution; mouse antiglomerular protein.” 15. Formalin solution, neutral buffered, 10 %.
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16. Specimen jars (100–200 mL). 17. ExtrAvidin-conjugated horseradish peroxidase peroxidase conjugated sheep anti-mouse IgG).
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18. Diaminobenzidine substrate tablet made up according to the manufacturer’s instructions.
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Methods
3.1 Preparation and Running of SDSPAGE Gel
Prepare two 10 % SDS-PAGE gels according to the manufacturer’s instructions for the SDS-PAGE platform being used. Please use gloves for all handing of acrylamide. Integrity of the gel assembly apparatus should be ensured before use (see Note 9). 1. In a falcon tube (15 mL), add 3.3 mL of 30 % acrylamide to 4.1 mL of water, 2.5 mL of 1.5 M Tris-Cl (pH 8.8), and 0.1 mL of 10 % SDS. After careful mixing and in a fume hood, add 50 µL of 10 % APS and 5 µL of TEMED and mix gently. This separating gel is added to the gel casting apparatus so as to form gels three quarters the final height of the gel. The unpolymerized gel is overlaid carefully with water (see Note 10) and left to polymerize for 20 min at room temperature (see Note 11). 2. Once the separating gel has polymerized, tilt the gel and remove the water from the top of gel (see Note 10). To a second falcon tube, add 5.4 mL of water, 2.0 mL of 30 % acrylamide solution, 2.5 mL of 0.5 M Tris–HCl, pH 6.8, and 1 mL of 10 % SDS. Add 10 % APS (50 µL) and TEMED (10 µL) and immediately add to the top of the separating gel. Insert wellforming comb according to the manufacturer’s instructions and allow to polymerize (approx. 20 min). Gels can be stored at this point (see Note 12). 3. Prepare samples of BSA, biotinylated-BSA, and bovine serum as described above (see Note 13). Add serum (20 µL) to 60 µL of sample buffer and centrifuge for 10 s at 14,000 × g at room temperature. Boil the samples for 5 min in a water bath and recentrifuge. Samples for loading are made by adding 15 µL of 350 µg/ mL of protein samples to 30 µL of sample buffer. Centrifuge as above, boil for 5 min, and recentrifuge (see Note 14). 4. Transfer gels to the running assembly and place into electrophoresis tanks filled with running buffer as per the manufacturer’s instructions. Remove well-forming combs and flush wells out with running buffer prior to loading boiled samples. 5. Load samples (1–5 µL) into wells. Prepare markers (Prestained or Kaleidoscope molecular mass markers for gels to be used for western blotting and unstained markers for Coomassie blue stained gels) and load onto gels according to the manufacturer’s
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Fig. 1 (a) Student generated Coomassie stained 10 % SDS-PAGE gel of 1 µL of BS containing 350 µg/B-BSA (lanes 1 and 2 ), 100 µg of BSA (lanes 3 and 4), 1 µL of BS labeled “Horse Serum” (lanes 5 and 6 ), and 100 µg of BSA labeled “Horse Serum Albumin” (lanes 7 and 8 ). (b) Western blot analysis for detection of B-BSA using alkaline phosphatase conjugated Extra-Avidin. 1 µL of BS containing 350 µg/mL B-BSA (lanes 1 and 2 ), 350 µg of B-BSA (labeled BSA) (Lanes 3 and 4 ), 1 µL of BS labeled “Horse Serum” (lanes 5 and 6 ), 350 ng BSA labeled “Horse Serum Albumin” (lanes 7 and 8 ). (Reproduced from [4] with permission from John Wiley and Sons)
instructions (see Note 15). Put on the lid and connect power pack and let electrophoresis run at 200 V for 1 h. 6. Let the bromophenol blue dye run just off the bottom of the gel and turn off power. Disassemble the assembly apparatus so that the gels are left sandwiched between the two glass plates. 7. Using a plastic wedge or spatula separate the plates which typically results in the gel being adhered to one of the plates. Transfer the gel into Coomassie blue stain by placing the gel and glass plate into a weigh boat containing the stain. Carefully remove the gel from the glass plate using the stain to wash the gel from the glass. Place the boat with the gel on a shaking platform for 20 min, after which decant the stain. Use a small amount of destain solution to wash the container and gel, removing excess dye. 8. Submerge the gel with fresh destain solution and place on the shaking platform (see Note 16). Replace the destain solution over a 2 h period or until the gel background becomes clear (see Fig. 1). 3.2
Western Blot
1. Remove gels for western blotting from the glass plates as above; however place into a container with transfer buffer on a shaking platform. Equilibrate gels with the buffer for at least 15 min before transfer. 2. In a shallow tray containing transfer buffer at 5 °C, place gauze pads (two per gel) and filter paper (two per gel) and leave to equilibrate for 5 min. At all times try and minimize entrapment of air bubbles into the material.
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3. Immerse the nitrocellulose membrane in 100 % methanol for 30 s and then transfer into a weigh boat filled with transfer buffer and place on a shaking platform/table. Equilibrate the membrane for 15 min before use. 4. Assemble the transfer cassette submerged in the shallow tray containing transfer buffer. The cassette is assembled consisting of a piece of gauze, filter paper, the gel, then the membrane, filter paper, and gauze. Roll a glass rod across the surface of the top layer of gauze while submerged to expel any air bubbles. Clamp shut the cassette and immediately transfer to an electroblotter apparatus filled with transfer buffer at 5 °C. As the orientation of the cassette is apparatus specific, this should be according the manufacturer’s instructions. Add the cooling unit and a magnetic stirring bar into the tank before placing the tank on a stirring plate, making sure there is agitation of the buffer. Attach lid with electrodes and run for 1 h at 100 V. 5. After 1 h, remove membranes from the cassette, and visually inspect for transfer of prestained markers. Using a soft lead pencil, mark these on the membrane as well as an outline of the position of the gel. It is expected that some high mass proteins may not transfer to the membrane completely, so maybe still evident in the gel. 6. Before the membranes have air-dried, transfer them into PBS or TBS and store at 5 °C until being ready for processing. 7. Transfer membranes into blocking solution (0.5 % (w/v) skim milk in TBS) and leave overnight at 5 °C. 8. To illustrate the process of a western blot, an optional step is to add a “primary antibody” to the antigen being detected. The primary antibody (1 µL of water added to the blocking solution), labeled “mouse monoclonal anti-BSA-IgG,” is not needed as the target protein is already biotinylated, which negates this step, if required (see Note 13). 9. For demonstration purposes, drain off the primary antibody and the wash the membrane further with 2 × 5 min of TBS. Drain the solution from the membrane. 10. Add the “secondary antibody” (labeled alkaline phosphatase conjugated goat anti-mouse IgG) ExtrAvidin-alkaline phosphatase diluted 1:20,000 (v/v) into the membrane and incubate for 1 h at room temperature on a shaking platform. Drain off the solution from the membrane, and wash further (2 × 5 min TBS) and drain before the addition of the substrate for alkaline phosphatase that will result in a precipitable colorimetric product. 11. Dissolve 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets in water as per the manufacturer’s instructions. Pour onto the membrane and agitate until color development is
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Fig. 2 Student generated dot blots for the detection and quantification of B-BSA in bovine serum. A1: 2 µL of BS containing 350 µg/mL B-BSA; A2: 2 µL BS containing 350 µg/mL B-BSA diluted 1 in 2; A3: 2 µL of BS containing 350 µg/mL B-BSA diluted 1 in 10; A4: 2 µL BS containing 350 µg/mL B-BSA diluted 1 in 50. B1: 2 µL of 350 µg/mL B-BSA; B2: 2 µL of 1 to 1 dilution of 350 µg/mL B-BSA; B3: 1 in 10 dilution of 350 µg/mL B-BSA; B4: 1 in 100 dilution of 350 µg/mL B-BSA. (Reproduced from [4] with permission from John Wiley and Sons)
apparent (see Note 17). Stop the reaction by removal of substrate and washing the membrane in water. Membranes can be air-dried and photographed (see Fig. 1b). 3.3
Dot Blots
1. Using an appropriate sized piece of nitrocellulose membrane (12 × 12 cm) draw a grid (1.5 cm squares) using a soft lead pencil. 2. After wetting the membrane with 100 % methanol, air-dry excess solvent from the membrane (see Note 18). Pipette the protein solutions (10 µL) described above (without sample buffer) directly onto the surface of the membrane. Samples can include positive controls, negative controls, and samples where the biotinylated protein content is diluted to contain 10–100 ng/10 µL of protein. Allow samples to sit on the membrane for 5 min for protein membrane adhesion before being adding into blocking buffer for at least 1 h. 3. Process the dot blots as per steps 8–11 in Subheading 3.2. Figure 2 shows a typical result for a dot blot experiment.
3.4 Immunohistochemistry (See Note 19)
1. Use surgical gloves and safety glasses through this whole procedure. 2. Dilute 0.2 mL of heparin with 0.8 mL saline to give 1,000 IU heparin in 1 mL of saline and place in 1 mL syringe with 20 gauge needle attached. 3. Dissolve 20 mg of biotin in 30 mL of saline (biotin-saline solution) and place in the 50 mL syringe with 20 gauge needle attached.
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4. Make up the anesthetic by dissolving 200 mg of ketamine in 1 mL of sterile water and dissolving 40 mg of xylazine in 1 mL of sterile water. Mix these together to give 2 mL of 100 mg/ mL ketamine and 20 mg/mL xylazine. 5. Fill the bottle from the perfusion set with 500 mL of formalin solution. Do this in a fume hood because formalin is toxic. 6. Still in the fume hood put the lid with the cannula back on the bottle and place the bottle upside down on its stand set a meter above the benchtop. At this stage have the needle at the end of the tube above the bottle so that the formalin solution does not leak out. 7. Place ~50 mL of formalin solution in the specimen jar. 8. Weigh the rat (see Note 20). 9. Inject 0.1 mL of the anesthetic per 100 g of body weight intraperitoneally, e.g., a 300 g rat will require 0.3 mL. 10. Once the rat is anesthetized (pinch hindfoot hard and if there is no response then you can proceed), open the chest cavity to reveal the heart. Using the 1 mL syringe slowly inject 1 mL of heparin saline into the left ventricle. 11. Remove the needle and then insert the needle of the 50 mL syringe into the left ventricle and cut a hole in the right atrium (snip with a pair of scissors). Slowly inject the 30 mL of the biotin-saline solution into the left ventricle. This will distribute the biotin through the blood vessels of the rat and allow escape of the excess fluid through the right atrium (see Note 21). 12. Remove the needle and insert the needle from the perfusion set into the left ventricle. Make sure that the formalin fixative is flowing from the needle before inserting into the ventricle. Note the rat will die at this stage and there is muscle contraction as the fixative reaches the muscles. This is normal. 13. Hold the needle in place (can be clamped with a paper clip) and allow the rat to be perfused with the 500 mL of fixative. 14. Remove the kidneys, cut the kidneys in half, and place in the formalin solution in the specimen jar for 24 h at 4 °C. 15. In the fume hood, pour off the formalin from the kidney pieces and replace it with saline. Gently mix for 1 min and then pour off the saline and replace with fresh saline. Allow to stand for at least 1 h and replace the saline again and allow to stand for at least 1 h. Can be left at 4 °C overnight at this stage. 16. Prepare the kidney pieces for wax embedding and embed in wax (see Note 22). This involves dehydrating the pieces through a series of alcohols at least 1 h in each (50 %, 70 %, 80 %, 90 %, 95 %, 100 %, 100 %) and then in three changes of
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xylene (20 min each). Place the pieces in melted wax (80 °C) three changes (1 h each) and embed in a block (see Notes 22 and 23). 17. Cut 7–10 µm sections using a microtome and place on glass microscope slides (see Note 23). 18. Place microscope slides with sections on a hotplate at 70 °C overnight (see Notes 24 and 25). 19. Dewax and rehydrate the sections. Place slides in three changes of xylenes (20 min each) and then rehydrate through a graded series of alcohols 5 min each step (100 %, 100 %, 95 %, 80 %, 70 %, 50 %, water, water). At this stage the sections are ready for immunohistochemistry. 20. A drop of 5 % serum is placed on the sections so that it covers the sections (~5 min). This is blocking serum to block nonantigenic sites. 21. Students make up the primary antibody into a 1:500 dilution with saline solution. 22. The drop of blocking serum is sucked off and drained from the sections and replaced by a drop of primary antibody solution (~20 min) (see Note 26). 23. The drop of primary antibody is sucked off and drained from the sections and replaced by a drop of blocking serum (~5 min). Repeat this step twice more. It is a washing step. 24. Incubate in “secondary antibody” ~20 min (see Note 27) which is really ExtrAvidin-conjugated horseradish peroxidase (labeled: peroxidase conjugated sheep anti-mouse IgG (see Note 28)). 25. The drop of secondary antibody is sucked off and drained from the sections and replaced by a drop of blocking serum (~5 min). Repeat this step twice more. This is a washing step. 26. A drop of diaminobenzidine solution is added to cover the section. 27. The section can then be observed under a microscope under low power to watch the reaction develop. 28. When the reaction is strong enough (glomeruli brown), the slide can be washed with saline. 29. For a class it is enough to then place a drop of saline on the section and put a coverslip on it. For more detailed view of the kidney structure and glomeruli, it should be counterstained with hematoxylin (stains the cell nuclei) dehydrated, cleared, and mounted under a coverslip. Typical result is shown in Fig. 3.
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Fig. 3 Pseudo-immunohistochemical result of kidney for the location of biotin after biotin perfusion. Note that the endothelial cells of kidney blood vessels are stained and the tubular epithelial cells are unstained. Scale = 100 µm (Reproduced from [4] with permission from John Wiley and Sons)
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Notes 1. Acrylamide is a known carcinogen. To reduce risk, commercially available premixed solutions are used. At all times acrylamide (both unpolymerized and polymerized) are handled while wearing gloves, lab coats, and safety glasses. All unpolymerized material is polymerized by the addition of catalysts (APS and TEMED) prior to disposal. 2. 10 % Ammonium persulfate needs to be made up fresh just before use. Poor quality APS is the most common reason why gels don’t polymerize. 3. Use TEMED in a fume hood and replace lid as soon as possible after opening. 4. Dithiothreitol (DTT) can be used instead of β-mercaptoethanol. These additives remove disulfide bonds in proteins. DTT can be used at a concentration of 500 mM in 3× sample buffer. 5. This protocol uses biotinylated-BSA which is commercially available. Other proteins can sourced either commercially or via conjugation of biotin to novel proteins using established protocols or commercial kits. 6. Can use either PVDF or nitrocellulose membrane. If using PVDF, note the membrane is hydrophobic and requires treatment with MeOH to allow wetting. The membrane should go semitransparent. During the course of membrane processing and sample application, do not let the membrane completely dry until the end of the experiment. Use gloves and forceps when handling the membranes. 7. You can use the cheaper phosphate buffered saline (PBS) instead of TBS in all steps except the final washings before
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addition of substrate. The phosphate in the buffer may act as an inhibitor of alkaline phosphatase. 8. Plastic weigh boats are ideal for the washing/stain steps of gels and membranes. Dimensions of the boats are such that they minimize volumes of reagents and are flexible enough to be reversibly deformed to facilitate pouring of solutions while allowing the gel/membrane to stay in the boat. 9. Prior to the addition of the gel solution, integrity of the gel assembly apparatus can be tested so that if leakage occurs it can be rectified before adding the unpolymerized acrylamide solution. This increases safety by not having to clean up spills of acrylamide, reduces wastage, and saves time. Using a squirt bottle containing 70 % ethanol fill the slot which forms the gel. Let stand for 5 min and monitor decreases in level of solvent in gel chamber. If no obvious leakage occurs, tilt the apparatus and remove the solvent. Insert the corner of a piece of paper towel to absorb any minor residual solvent. The casting assembly can then be used for gel polymerization. 10. Overlaying of water after addition of resolving gel usually requires demonstration to novices. As DI water is less dense than the acrylamide solution, it will overlay the gel prior to polymerization. However care is needed as violent addition of the water will cause mixing with the gel, thus causing disruption and mixing with the resolving gel. To overlay the gel, use a 200 µL capacity pipette filled with DI water and carefully dispense the water down the side wall of the reservoir holding the gel in a smooth and gentle manner. Repeat placing the water down the other side wall. You can see the water overlaying the gel due to the differences in refractive index of the two solutions. Make sure that the two additions of water meet in the middle of the gel. After polymerization, tilting the apparatus will reveal a layer of water on top of the solid gel. Remove the liquid using a paper towel to draw up the solution by capillary action. The gel is now ready for addition of the stacking gel. 11. If any acrylamide solution is left after filling the gel casting apparatus, use it to visually monitor the polymerization process by watching for solidification. 12. Gels can be stored for up to 2 weeks at 5 °C. Remove glass plates containing gels from the clamping cassette, wrap in paper towel which has been saturated with running buffer, and place into a sealed plastic container. Store at 5 °C until use. 13. A variety of experiments can be designed using these samples. In trying to demonstrate blotting, the use of a primary antibody is indicated to simulate a typical western blot. A full description of the use of biotin in an undergraduate immunology class is found in ref. 4.
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14. The use of the centrifuge is to make sure that samples are appropriately mixed and located in the tube. When boiling samples prior to loading onto gels, microtubes will pop their lids unless either a lid locking device is used or the lids are punctured prior to boiling. Typically we use a syringe needle to pierce the lids and place the tubes into floating foam tube holder. Centrifuge tubes before loading onto gel. 15. There are a range of markers available from many manufactures. Prestained markers are expensive but it means that protein separation can be monitored in real time while the gel is running. In addition these markers can be used to assess the success of the electroblotting step and to indicate the position of size marker proteins on membranes. 16. To reduce the amount of destain solution used, tissues (e.g., Kimwipes and other brands) or pieces of foam rubber (approx. 1.5 cm × 1.5 cm, need to test by trial and error) can be added to the destain solution while the gel is destaining. These materials selectively bind Coomassie blue and reduce the need for repeated destain exchange. Please note some materials can completely destain the gel (including proteins) if left for extended lengths of time. 17. Activity of the conjugated enzyme can be tested by adding 1 µL of the diluted ExtrAvidin-alkaline phosphatase to 1 mL of substrate solution. The enzyme should turn the solution purple. 18. Partially air-dry membrane so as the addition of fluid drops to the membrane does not cause excess spreading. 19. Anesthetizing and perfusion of a rat requires expertise. It might be better to approach a research institute, hospital, or university to carry out this procedure for you. It is standard practice in many research institutes. 20. It is important to weigh the rat just prior to anesthesia for calculating the anesthetic dose as rats can change their weight considerably over a day. 21. The biotin sticks to the glomeruli in the kidneys during this process and thus labeling them. 22. Dehydrating, embedding, and sectioning tissue requires expertise and generally animal ethics approval. It might be better to approach a research institute, hospital, or university to carry out this procedure for you. It is standard practice in many research institutes. 23. Once in a wax block the tissue can be kept for centuries. 24. This is important because it adheres to the sections to the slides so that they do not wash off during subsequent processing. 25. Wax sections on a glass slide can be kept for centuries.
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26. Again this step can be omitted but is included when trying to simulate the need for a primary antibody. This is fake primary antibody because what will ultimately be detected is the biotin which is bound to the glomeruli in the perfusion of the rat. 27. The time can be shortened or lengthened to suit your practical class because of the primary antibody step being simulated. 28. The working concentration of ExtrAvidin-Peroxidase is 1:100 (v/v). It is suggested that the technical staff make it up into the working dilution rather than the students. This will save mistakes and money. References 1. Diamandis EP, Christopoulos TK (1991) The Biotin-(Strept)Avidin System: principles and applications in biotechnology. Clin Chem 37:625–636 2. Gorst J, Lee S (2005) The undergraduate life sciences laboratory: student expectations, approaches to learning and implications for teaching. In: Teaching in the sciences: learner-centred approaches. The Haworth Press Inc, New York
3. Agus HM (2010) Significance of laboratory experience in undergraduate microbiology. Microbiol. Australia, March, 38–40 4. Millar T, Knighton R, Chuck J (2012) The use of biotin to demonstrate immunohistochemistry, western blotting, and dot blots in university practical classes. Biochem Mol Biol Educ 40: 246–253
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Introduction The immunodetection methods outlined in this chapter exploit the affinity between the small molecule biotin (vitamin B7) and the protein avidin. Biotin is a naturally occurring molecule typically made by the microbiota in the human gut and is an important co-factor in many biochemical reactions. However biotin is used extensively in biotechnology in immunodetection processes [1]. Exploiting the very high affinity of the molecule to avidin, biotin-avidin-enzyme complexes can be detected using substrates that produce colorimetric products. This reporting system is commonly used in blots, immunohistochemistry, and ELISAs. In this study, the molecule is exogenously added to complex biological samples for detection to demonstrate these modern immunological methods. As the selectivity and detection of molecule–ligand interactions is used in a range of different biology laboratories, it is a very important technique to demonstrate to undergraduate science students [2, 3]. Beyond its use in diagnostics, the technique is
Biji T. Kurien and R. Hal Scofield (eds.), Detection of Blotted Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 1314, DOI 10.1007/978-1-4939-2718-0_16, © Springer Science+Business Media New York 2015
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often central to many protein chemistry, histology, and cell biology research projects. Both medical and biological science undergraduate students need to develop skills for carrying out these common methodologies. However, for higher education institutions, running practical classes teaching these skills is almost impossible because of the financial burden, time constraints, resource limitations, and obtaining a realistic outcome within the hands of novices [2, 3]. To accommodate students handling small volumes of very expensive reagents in experiments that take longer than a typical 4 h undergraduate laboratory, sessions often become mainly demonstrations, with key steps illustrated by prepared samples. Using the detection of biotin to illustrate this process, crucial steps such as incubations, blocking, and washing with shorter time periods are possible without the risk of interfering with the quality of the results obtained. Having a failsafe technique where the outcome is assured means that the educational process could be freed up to transfer ownership of the practical to the students [4].
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Materials Prepare all reagents using deionized water.
2.1 Gel Electrophoresis
1. Gel electrophoresis apparatus: There are many available with slightly different formats, e.g., Mini-Protean III (BioRad Herclues, CA, USA). 2. Other specialist equipment: Microcentrifuge, shaking or rocking platform, magnetic stirrer, boiling water bath, power packs, micro-pipettes, microcentrifuge tubes and racks (including those that float in water baths). 3. Resolving gel buffer: 1.5 M Tris–HCl (pH 8.8): Weigh out 18.2 g Tris base and dissolve in 80 mL water using a stirrer. Adjust to pH 8.8 with concentrated HCl and make up to 100 mL with water. Store at 4 °C (need approx. 3 mL per gel). 4. Stacking gel buffer: 0.5 M Tris–HCl (pH 6.8): Weigh 6 g Tris base and dissolve in 60 mL water using a stirrer. Adjust to pH 6.8 with concentrated HCl and make up to 100 mL with water. Store at 4 °C (need approx. 3 mL per gel). 5. 10 % (w/v) SDS: Using a balance in fume hood, weigh out 10 g of SDS. Make up to 100 mL using water. Store at room temperature (need 200 µL per gel). 6. 30 % acrylamide/Bis solution (29:1 acrylamide: Bis, 3.3 % cross-linker concentration): purchase premixed (BioRad Hercules, CA USA), store at 4 °C (need approx. 4 mL per gel) (see Note 1). 7. 10 % (w/v) ammonium persulfate: Weigh out 1 g and make up to 10 mL with water. Store at room temperature (need 100 µL per gel) (see Note 2).
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8. N,N,N′,N′-tetramethyl-ethylenediamine (TEMED): Store at 4 °C (see Note 3) (need 30 µL per gel). 9. SDS-PAGE running buffer: 0.025 M Tris–HCl, pH 8.3, 0.192 M glycine, 0.1 % SDS. To make a 10× stock, weigh out 30 g of Tris base, 144 g of glycine, 1 g of SDS and make up to 1 L with water (pH should not have to be adjusted). Dilute one part in ten in water before use. 10. Sample buffer: Add water (3.55 mL), 0.5 M Tris–HCl, pH 6.8 (1.25 mL), glycerol (2.5 mL), 10 % SDS (2.0 mL), and bromophenol blue (0.2 mL of a 0.5 % (w/v) solution in water). Store at room temperature. Add 50 µL of mercaptoethanol to 950 µL of sample buffer just before use (see Note 4). 11. Coomassie blue stain: 0.1 % (w/v) Coomassie blue in 40 % (v/v) methanol, 10 % (v/v) acetic acid, and 50 % (v/v) water. 12. Destaining solution: Add concentrated acetic acid (250 mL), methanol (1 L), and water (1,250 mL). 13. Protein molecular mass markers: Many are available including Precision Plus Protein Kaleidoscope Standards 10–250 kDa, SDS-PAGE Broad range Standards (unstained), (6.5–200 kDa). 14. 70 % (v/v) ethanol. 15. Samples: (a) Bovine serum (or horse serum) with 350 µg/mL biotinylated bovine serum albumin (B-BSA) (see Note 5). Weigh out 2.5 mg of B-BSA and make up to 250 µL with water. Add 35 µL to 1 mL of serum. (b) Bovine serum albumin (BSA, 0.5 mg/mL): Weigh out 5 mg of BSA and dissolve in 10 mL of water. (c) B-BSA (350 µg/mL). Take 35 µL of the 10 mg/mL solution of B-BSA and make up to 1 mL with water. (d) Negative control serum: use bovine serum (or horse serum). 2.2 Western Blotting and Immunoblots
1. Wet protein transfer apparatus: Many are available, e.g., Mini Trans-Blot (BioRad Hercules, CA USA). These will include gauze pads as support for the gel transfer assembly. Fill the cooling units with water and store at −20 °C. 2. PVDF membrane (one per gel). Cut to the size of gel (see Note 6). 3. Filter paper (two per gel) cut to the same size as gauze pads. 4. Transfer buffer: 25 mM Tris, pH 8.3, 192 mM glycine, 20 % (v/v) methanol, and 0.5 % SDS. Weigh out, Tris Base 3 g/L, glycine 14.4 g/L, methanol 200 mL/L, SDS 5 g/L and make up to 1 L with water. Do not adjust pH. Store and use at 5 °C. Approx. 1 L of buffer per tank is required.
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5. Tris Buffered Saline (TBS): 50 mM Tris–HCl pH 7.4, 150 mM NaCl (see Note 7). Typically this is made up as a 5× stock which is diluted before use. Weigh out 30.2 g Tris and 43.5 g of NaCl and dissolve in 800 mL of water. Adjust pH to 7.4 using concentrated HCl. Make up to 1 L. Before use, dilute one part in four using water. 6. Blocking solution: 5 % (w/v) skim milk power in TBS (see Note 7). Store at 4 °C. 7. Square plastic weigh boats approx. 15 cm × 2.5 cm and a shallow tray approx. 40 × 20 × 10 cm (see Note 8). 8. Glass rod (20 cm × 0.5 cm). 9. SIGMA-FAST BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium) tablets. 10. ExtrAvidin-alkaline phosphatase. 2.3 Immunohistochemistry
1. One mature rat. Strain is not important. 2. S-(+)-Ketamine hydrochloride. 3. Xylazine hydrochloride. 4. Ethanol. 5. Xylenes. 6. Paraplast wax for histology. 7. Sterile water (Note: Since this procedure is nonrecovery anesthesia, boiled water would suffice). 8. 5,000 IU/mL heparin. 9. Saline: Make up 1 L of saline by adding 9 g of NaCl to 1 L of sterile water and mixing. 10. Perfusion set and stand: This is a bottle (1 L) that can be hung upside down on a stand 1 m above the animal to be perfused. A hole is made in the lid of the bottle to allow a 5 mm diameter cannula to be poked through the lid so that it does not leak. A ~2 m tube is attached to the free end of cannula and a 16 gauge needle is attached to this for inserting into the left ventricle of the rat heart. 11. Biotin (100 mg). 12. Syringes: 1 and 50 mL with 20 gauge needles. 13. 5 % serum: Take 5 mL of bovine serum and dilute it with 95 mL of saline solution. 14. Primary antibody stock solution: 0.5 mL of bovine serum with 9.5 mL of saline solution. About 1 mL is put into a vial and labeled “stock primary antibody solution; mouse antiglomerular protein.” 15. Formalin solution, neutral buffered, 10 %.
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16. Specimen jars (100–200 mL). 17. ExtrAvidin-conjugated horseradish peroxidase peroxidase conjugated sheep anti-mouse IgG).
(labeled:
18. Diaminobenzidine substrate tablet made up according to the manufacturer’s instructions.
3
Methods
3.1 Preparation and Running of SDSPAGE Gel
Prepare two 10 % SDS-PAGE gels according to the manufacturer’s instructions for the SDS-PAGE platform being used. Please use gloves for all handing of acrylamide. Integrity of the gel assembly apparatus should be ensured before use (see Note 9). 1. In a falcon tube (15 mL), add 3.3 mL of 30 % acrylamide to 4.1 mL of water, 2.5 mL of 1.5 M Tris-Cl (pH 8.8), and 0.1 mL of 10 % SDS. After careful mixing and in a fume hood, add 50 µL of 10 % APS and 5 µL of TEMED and mix gently. This separating gel is added to the gel casting apparatus so as to form gels three quarters the final height of the gel. The unpolymerized gel is overlaid carefully with water (see Note 10) and left to polymerize for 20 min at room temperature (see Note 11). 2. Once the separating gel has polymerized, tilt the gel and remove the water from the top of gel (see Note 10). To a second falcon tube, add 5.4 mL of water, 2.0 mL of 30 % acrylamide solution, 2.5 mL of 0.5 M Tris–HCl, pH 6.8, and 1 mL of 10 % SDS. Add 10 % APS (50 µL) and TEMED (10 µL) and immediately add to the top of the separating gel. Insert wellforming comb according to the manufacturer’s instructions and allow to polymerize (approx. 20 min). Gels can be stored at this point (see Note 12). 3. Prepare samples of BSA, biotinylated-BSA, and bovine serum as described above (see Note 13). Add serum (20 µL) to 60 µL of sample buffer and centrifuge for 10 s at 14,000 × g at room temperature. Boil the samples for 5 min in a water bath and recentrifuge. Samples for loading are made by adding 15 µL of 350 µg/ mL of protein samples to 30 µL of sample buffer. Centrifuge as above, boil for 5 min, and recentrifuge (see Note 14). 4. Transfer gels to the running assembly and place into electrophoresis tanks filled with running buffer as per the manufacturer’s instructions. Remove well-forming combs and flush wells out with running buffer prior to loading boiled samples. 5. Load samples (1–5 µL) into wells. Prepare markers (Prestained or Kaleidoscope molecular mass markers for gels to be used for western blotting and unstained markers for Coomassie blue stained gels) and load onto gels according to the manufacturer’s
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Fig. 1 (a) Student generated Coomassie stained 10 % SDS-PAGE gel of 1 µL of BS containing 350 µg/B-BSA (lanes 1 and 2 ), 100 µg of BSA (lanes 3 and 4), 1 µL of BS labeled “Horse Serum” (lanes 5 and 6 ), and 100 µg of BSA labeled “Horse Serum Albumin” (lanes 7 and 8 ). (b) Western blot analysis for detection of B-BSA using alkaline phosphatase conjugated Extra-Avidin. 1 µL of BS containing 350 µg/mL B-BSA (lanes 1 and 2 ), 350 µg of B-BSA (labeled BSA) (Lanes 3 and 4 ), 1 µL of BS labeled “Horse Serum” (lanes 5 and 6 ), 350 ng BSA labeled “Horse Serum Albumin” (lanes 7 and 8 ). (Reproduced from [4] with permission from John Wiley and Sons)
instructions (see Note 15). Put on the lid and connect power pack and let electrophoresis run at 200 V for 1 h. 6. Let the bromophenol blue dye run just off the bottom of the gel and turn off power. Disassemble the assembly apparatus so that the gels are left sandwiched between the two glass plates. 7. Using a plastic wedge or spatula separate the plates which typically results in the gel being adhered to one of the plates. Transfer the gel into Coomassie blue stain by placing the gel and glass plate into a weigh boat containing the stain. Carefully remove the gel from the glass plate using the stain to wash the gel from the glass. Place the boat with the gel on a shaking platform for 20 min, after which decant the stain. Use a small amount of destain solution to wash the container and gel, removing excess dye. 8. Submerge the gel with fresh destain solution and place on the shaking platform (see Note 16). Replace the destain solution over a 2 h period or until the gel background becomes clear (see Fig. 1). 3.2
Western Blot
1. Remove gels for western blotting from the glass plates as above; however place into a container with transfer buffer on a shaking platform. Equilibrate gels with the buffer for at least 15 min before transfer. 2. In a shallow tray containing transfer buffer at 5 °C, place gauze pads (two per gel) and filter paper (two per gel) and leave to equilibrate for 5 min. At all times try and minimize entrapment of air bubbles into the material.
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3. Immerse the nitrocellulose membrane in 100 % methanol for 30 s and then transfer into a weigh boat filled with transfer buffer and place on a shaking platform/table. Equilibrate the membrane for 15 min before use. 4. Assemble the transfer cassette submerged in the shallow tray containing transfer buffer. The cassette is assembled consisting of a piece of gauze, filter paper, the gel, then the membrane, filter paper, and gauze. Roll a glass rod across the surface of the top layer of gauze while submerged to expel any air bubbles. Clamp shut the cassette and immediately transfer to an electroblotter apparatus filled with transfer buffer at 5 °C. As the orientation of the cassette is apparatus specific, this should be according the manufacturer’s instructions. Add the cooling unit and a magnetic stirring bar into the tank before placing the tank on a stirring plate, making sure there is agitation of the buffer. Attach lid with electrodes and run for 1 h at 100 V. 5. After 1 h, remove membranes from the cassette, and visually inspect for transfer of prestained markers. Using a soft lead pencil, mark these on the membrane as well as an outline of the position of the gel. It is expected that some high mass proteins may not transfer to the membrane completely, so maybe still evident in the gel. 6. Before the membranes have air-dried, transfer them into PBS or TBS and store at 5 °C until being ready for processing. 7. Transfer membranes into blocking solution (0.5 % (w/v) skim milk in TBS) and leave overnight at 5 °C. 8. To illustrate the process of a western blot, an optional step is to add a “primary antibody” to the antigen being detected. The primary antibody (1 µL of water added to the blocking solution), labeled “mouse monoclonal anti-BSA-IgG,” is not needed as the target protein is already biotinylated, which negates this step, if required (see Note 13). 9. For demonstration purposes, drain off the primary antibody and the wash the membrane further with 2 × 5 min of TBS. Drain the solution from the membrane. 10. Add the “secondary antibody” (labeled alkaline phosphatase conjugated goat anti-mouse IgG) ExtrAvidin-alkaline phosphatase diluted 1:20,000 (v/v) into the membrane and incubate for 1 h at room temperature on a shaking platform. Drain off the solution from the membrane, and wash further (2 × 5 min TBS) and drain before the addition of the substrate for alkaline phosphatase that will result in a precipitable colorimetric product. 11. Dissolve 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets in water as per the manufacturer’s instructions. Pour onto the membrane and agitate until color development is
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Fig. 2 Student generated dot blots for the detection and quantification of B-BSA in bovine serum. A1: 2 µL of BS containing 350 µg/mL B-BSA; A2: 2 µL BS containing 350 µg/mL B-BSA diluted 1 in 2; A3: 2 µL of BS containing 350 µg/mL B-BSA diluted 1 in 10; A4: 2 µL BS containing 350 µg/mL B-BSA diluted 1 in 50. B1: 2 µL of 350 µg/mL B-BSA; B2: 2 µL of 1 to 1 dilution of 350 µg/mL B-BSA; B3: 1 in 10 dilution of 350 µg/mL B-BSA; B4: 1 in 100 dilution of 350 µg/mL B-BSA. (Reproduced from [4] with permission from John Wiley and Sons)
apparent (see Note 17). Stop the reaction by removal of substrate and washing the membrane in water. Membranes can be air-dried and photographed (see Fig. 1b). 3.3
Dot Blots
1. Using an appropriate sized piece of nitrocellulose membrane (12 × 12 cm) draw a grid (1.5 cm squares) using a soft lead pencil. 2. After wetting the membrane with 100 % methanol, air-dry excess solvent from the membrane (see Note 18). Pipette the protein solutions (10 µL) described above (without sample buffer) directly onto the surface of the membrane. Samples can include positive controls, negative controls, and samples where the biotinylated protein content is diluted to contain 10–100 ng/10 µL of protein. Allow samples to sit on the membrane for 5 min for protein membrane adhesion before being adding into blocking buffer for at least 1 h. 3. Process the dot blots as per steps 8–11 in Subheading 3.2. Figure 2 shows a typical result for a dot blot experiment.
3.4 Immunohistochemistry (See Note 19)
1. Use surgical gloves and safety glasses through this whole procedure. 2. Dilute 0.2 mL of heparin with 0.8 mL saline to give 1,000 IU heparin in 1 mL of saline and place in 1 mL syringe with 20 gauge needle attached. 3. Dissolve 20 mg of biotin in 30 mL of saline (biotin-saline solution) and place in the 50 mL syringe with 20 gauge needle attached.
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4. Make up the anesthetic by dissolving 200 mg of ketamine in 1 mL of sterile water and dissolving 40 mg of xylazine in 1 mL of sterile water. Mix these together to give 2 mL of 100 mg/ mL ketamine and 20 mg/mL xylazine. 5. Fill the bottle from the perfusion set with 500 mL of formalin solution. Do this in a fume hood because formalin is toxic. 6. Still in the fume hood put the lid with the cannula back on the bottle and place the bottle upside down on its stand set a meter above the benchtop. At this stage have the needle at the end of the tube above the bottle so that the formalin solution does not leak out. 7. Place ~50 mL of formalin solution in the specimen jar. 8. Weigh the rat (see Note 20). 9. Inject 0.1 mL of the anesthetic per 100 g of body weight intraperitoneally, e.g., a 300 g rat will require 0.3 mL. 10. Once the rat is anesthetized (pinch hindfoot hard and if there is no response then you can proceed), open the chest cavity to reveal the heart. Using the 1 mL syringe slowly inject 1 mL of heparin saline into the left ventricle. 11. Remove the needle and then insert the needle of the 50 mL syringe into the left ventricle and cut a hole in the right atrium (snip with a pair of scissors). Slowly inject the 30 mL of the biotin-saline solution into the left ventricle. This will distribute the biotin through the blood vessels of the rat and allow escape of the excess fluid through the right atrium (see Note 21). 12. Remove the needle and insert the needle from the perfusion set into the left ventricle. Make sure that the formalin fixative is flowing from the needle before inserting into the ventricle. Note the rat will die at this stage and there is muscle contraction as the fixative reaches the muscles. This is normal. 13. Hold the needle in place (can be clamped with a paper clip) and allow the rat to be perfused with the 500 mL of fixative. 14. Remove the kidneys, cut the kidneys in half, and place in the formalin solution in the specimen jar for 24 h at 4 °C. 15. In the fume hood, pour off the formalin from the kidney pieces and replace it with saline. Gently mix for 1 min and then pour off the saline and replace with fresh saline. Allow to stand for at least 1 h and replace the saline again and allow to stand for at least 1 h. Can be left at 4 °C overnight at this stage. 16. Prepare the kidney pieces for wax embedding and embed in wax (see Note 22). This involves dehydrating the pieces through a series of alcohols at least 1 h in each (50 %, 70 %, 80 %, 90 %, 95 %, 100 %, 100 %) and then in three changes of
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xylene (20 min each). Place the pieces in melted wax (80 °C) three changes (1 h each) and embed in a block (see Notes 22 and 23). 17. Cut 7–10 µm sections using a microtome and place on glass microscope slides (see Note 23). 18. Place microscope slides with sections on a hotplate at 70 °C overnight (see Notes 24 and 25). 19. Dewax and rehydrate the sections. Place slides in three changes of xylenes (20 min each) and then rehydrate through a graded series of alcohols 5 min each step (100 %, 100 %, 95 %, 80 %, 70 %, 50 %, water, water). At this stage the sections are ready for immunohistochemistry. 20. A drop of 5 % serum is placed on the sections so that it covers the sections (~5 min). This is blocking serum to block nonantigenic sites. 21. Students make up the primary antibody into a 1:500 dilution with saline solution. 22. The drop of blocking serum is sucked off and drained from the sections and replaced by a drop of primary antibody solution (~20 min) (see Note 26). 23. The drop of primary antibody is sucked off and drained from the sections and replaced by a drop of blocking serum (~5 min). Repeat this step twice more. It is a washing step. 24. Incubate in “secondary antibody” ~20 min (see Note 27) which is really ExtrAvidin-conjugated horseradish peroxidase (labeled: peroxidase conjugated sheep anti-mouse IgG (see Note 28)). 25. The drop of secondary antibody is sucked off and drained from the sections and replaced by a drop of blocking serum (~5 min). Repeat this step twice more. This is a washing step. 26. A drop of diaminobenzidine solution is added to cover the section. 27. The section can then be observed under a microscope under low power to watch the reaction develop. 28. When the reaction is strong enough (glomeruli brown), the slide can be washed with saline. 29. For a class it is enough to then place a drop of saline on the section and put a coverslip on it. For more detailed view of the kidney structure and glomeruli, it should be counterstained with hematoxylin (stains the cell nuclei) dehydrated, cleared, and mounted under a coverslip. Typical result is shown in Fig. 3.
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Fig. 3 Pseudo-immunohistochemical result of kidney for the location of biotin after biotin perfusion. Note that the endothelial cells of kidney blood vessels are stained and the tubular epithelial cells are unstained. Scale = 100 µm (Reproduced from [4] with permission from John Wiley and Sons)
4
Notes 1. Acrylamide is a known carcinogen. To reduce risk, commercially available premixed solutions are used. At all times acrylamide (both unpolymerized and polymerized) are handled while wearing gloves, lab coats, and safety glasses. All unpolymerized material is polymerized by the addition of catalysts (APS and TEMED) prior to disposal. 2. 10 % Ammonium persulfate needs to be made up fresh just before use. Poor quality APS is the most common reason why gels don’t polymerize. 3. Use TEMED in a fume hood and replace lid as soon as possible after opening. 4. Dithiothreitol (DTT) can be used instead of β-mercaptoethanol. These additives remove disulfide bonds in proteins. DTT can be used at a concentration of 500 mM in 3× sample buffer. 5. This protocol uses biotinylated-BSA which is commercially available. Other proteins can sourced either commercially or via conjugation of biotin to novel proteins using established protocols or commercial kits. 6. Can use either PVDF or nitrocellulose membrane. If using PVDF, note the membrane is hydrophobic and requires treatment with MeOH to allow wetting. The membrane should go semitransparent. During the course of membrane processing and sample application, do not let the membrane completely dry until the end of the experiment. Use gloves and forceps when handling the membranes. 7. You can use the cheaper phosphate buffered saline (PBS) instead of TBS in all steps except the final washings before
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addition of substrate. The phosphate in the buffer may act as an inhibitor of alkaline phosphatase. 8. Plastic weigh boats are ideal for the washing/stain steps of gels and membranes. Dimensions of the boats are such that they minimize volumes of reagents and are flexible enough to be reversibly deformed to facilitate pouring of solutions while allowing the gel/membrane to stay in the boat. 9. Prior to the addition of the gel solution, integrity of the gel assembly apparatus can be tested so that if leakage occurs it can be rectified before adding the unpolymerized acrylamide solution. This increases safety by not having to clean up spills of acrylamide, reduces wastage, and saves time. Using a squirt bottle containing 70 % ethanol fill the slot which forms the gel. Let stand for 5 min and monitor decreases in level of solvent in gel chamber. If no obvious leakage occurs, tilt the apparatus and remove the solvent. Insert the corner of a piece of paper towel to absorb any minor residual solvent. The casting assembly can then be used for gel polymerization. 10. Overlaying of water after addition of resolving gel usually requires demonstration to novices. As DI water is less dense than the acrylamide solution, it will overlay the gel prior to polymerization. However care is needed as violent addition of the water will cause mixing with the gel, thus causing disruption and mixing with the resolving gel. To overlay the gel, use a 200 µL capacity pipette filled with DI water and carefully dispense the water down the side wall of the reservoir holding the gel in a smooth and gentle manner. Repeat placing the water down the other side wall. You can see the water overlaying the gel due to the differences in refractive index of the two solutions. Make sure that the two additions of water meet in the middle of the gel. After polymerization, tilting the apparatus will reveal a layer of water on top of the solid gel. Remove the liquid using a paper towel to draw up the solution by capillary action. The gel is now ready for addition of the stacking gel. 11. If any acrylamide solution is left after filling the gel casting apparatus, use it to visually monitor the polymerization process by watching for solidification. 12. Gels can be stored for up to 2 weeks at 5 °C. Remove glass plates containing gels from the clamping cassette, wrap in paper towel which has been saturated with running buffer, and place into a sealed plastic container. Store at 5 °C until use. 13. A variety of experiments can be designed using these samples. In trying to demonstrate blotting, the use of a primary antibody is indicated to simulate a typical western blot. A full description of the use of biotin in an undergraduate immunology class is found in ref. 4.
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14. The use of the centrifuge is to make sure that samples are appropriately mixed and located in the tube. When boiling samples prior to loading onto gels, microtubes will pop their lids unless either a lid locking device is used or the lids are punctured prior to boiling. Typically we use a syringe needle to pierce the lids and place the tubes into floating foam tube holder. Centrifuge tubes before loading onto gel. 15. There are a range of markers available from many manufactures. Prestained markers are expensive but it means that protein separation can be monitored in real time while the gel is running. In addition these markers can be used to assess the success of the electroblotting step and to indicate the position of size marker proteins on membranes. 16. To reduce the amount of destain solution used, tissues (e.g., Kimwipes and other brands) or pieces of foam rubber (approx. 1.5 cm × 1.5 cm, need to test by trial and error) can be added to the destain solution while the gel is destaining. These materials selectively bind Coomassie blue and reduce the need for repeated destain exchange. Please note some materials can completely destain the gel (including proteins) if left for extended lengths of time. 17. Activity of the conjugated enzyme can be tested by adding 1 µL of the diluted ExtrAvidin-alkaline phosphatase to 1 mL of substrate solution. The enzyme should turn the solution purple. 18. Partially air-dry membrane so as the addition of fluid drops to the membrane does not cause excess spreading. 19. Anesthetizing and perfusion of a rat requires expertise. It might be better to approach a research institute, hospital, or university to carry out this procedure for you. It is standard practice in many research institutes. 20. It is important to weigh the rat just prior to anesthesia for calculating the anesthetic dose as rats can change their weight considerably over a day. 21. The biotin sticks to the glomeruli in the kidneys during this process and thus labeling them. 22. Dehydrating, embedding, and sectioning tissue requires expertise and generally animal ethics approval. It might be better to approach a research institute, hospital, or university to carry out this procedure for you. It is standard practice in many research institutes. 23. Once in a wax block the tissue can be kept for centuries. 24. This is important because it adheres to the sections to the slides so that they do not wash off during subsequent processing. 25. Wax sections on a glass slide can be kept for centuries.
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26. Again this step can be omitted but is included when trying to simulate the need for a primary antibody. This is fake primary antibody because what will ultimately be detected is the biotin which is bound to the glomeruli in the perfusion of the rat. 27. The time can be shortened or lengthened to suit your practical class because of the primary antibody step being simulated. 28. The working concentration of ExtrAvidin-Peroxidase is 1:100 (v/v). It is suggested that the technical staff make it up into the working dilution rather than the students. This will save mistakes and money. References 1. Diamandis EP, Christopoulos TK (1991) The Biotin-(Strept)Avidin System: principles and applications in biotechnology. Clin Chem 37:625–636 2. Gorst J, Lee S (2005) The undergraduate life sciences laboratory: student expectations, approaches to learning and implications for teaching. In: Teaching in the sciences: learner-centred approaches. The Haworth Press Inc, New York
3. Agus HM (2010) Significance of laboratory experience in undergraduate microbiology. Microbiol. Australia, March, 38–40 4. Millar T, Knighton R, Chuck J (2012) The use of biotin to demonstrate immunohistochemistry, western blotting, and dot blots in university practical classes. Biochem Mol Biol Educ 40: 246–253
