CHAPTER SIXTEEN
Protein Filter Binding Sarah Kolitz*,1, Jon R. Lorsch†
*Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA † Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 4.3 Tip 4.4 Caution 5. Step 1 Assemble Binding Reactions 5.1 Overview 5.2 Duration 5.3 Tip 5.4 Tip 5.5 Tip 5.6 Tip 5.7 Tip 6. Step 2 Quantify Binding 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 6.5 Tip 7. Step 3 Process Binding Data 7.1 Overview 7.2 Duration References Source References
198 198 198 199 199 199 200 200 200 201 201 201 201 201 201 201 202 202 202 202 203 203 203 204 204 204 205 205
Abstract This protocol describes a method to monitor the binding of nucleic acid to protein, allowing the determination of the apparent affinity of a nucleic acid–protein interaction. Methods in Enzymology, Volume 541 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420119-4.00016-1
#
2014 Elsevier Inc. All rights reserved.
197
198
Sarah Kolitz and Jon R. Lorsch
1. THEORY This method relies on the fact that most proteins will bind to a nitrocellulose filter, while free nucleic acid passes through. Thus if a radiolabeled nucleic acid is bound to a protein, it can be detected on the filter, whereas unbound nucleic acid will pass through (Nirenberg and Leder, 1964). In this way, a sequence of points at different protein concentrations or times can be observed in order to obtain thermodynamic binding curves or kinetic time courses for the interaction between the protein and nucleic acid. Since each concentration (or time) point is done using a separate filter, variability due to pipetting errors in the total amount of material added to each filter can contribute to noise in the data. This can be addressed by adding a filter that binds nucleic acid (such as Nytran™ SuPerCharge (Whatman)) below the nitrocellulose filter, such that variability in the amount of labeled nucleic acid loaded onto the filter can be taken into account (Wong and Lohman, 1993). Advantages of this technique include its relative ease and speed as well as the fact that it does not require particularly expensive equipment. Some proteins, however, bind poorly to nitrocellulose, or may be unable to bind nitrocellulose and nucleic acid simultaneously (Oehler et al., 1999); thus in certain situations this technique cannot be applied. In addition, if the rate of dissociation of the complex is high, it may fall apart during the washing phase of the protocol. In these cases, alternative approaches to monitoring binding must be undertaken, including native gel electrophoresis and fluorescence anisotropy.
2. EQUIPMENT Vacuum manifold Scintillation counter Micropipettors (P1000 and P20) Micropipettor tips 1.5-ml microcentrifuge tubes Whatman GF/C filters (24 mm) Nytran™ SuPerCharge (SPC) membranes
3. MATERIALS Radiolabeled nucleic acid of interest Protein of interest
199
Protein Filter Binding
HEPES Potassium hydroxide (KOH) Magnesium acetate (MgOAc) Potassium acetate (KOAc) Glacial acetic acid Glycerol Dithiothreitol (DTT) Creatine kinase Scintillation fluid
3.1. Solutions & buffers Step 1 Protein dilution buffer (for eIF2) Component
Final concentration
Stock
Amount
HEPES–KOH, pH 7.6
20 mM
1M
20 ml
KOAc, pH 7.6
100 mM
1M
100 ml
MgOAc
0.1 mM
1M
0.1 ml
Glycerol
10%
100%
100 ml
DTT*
2 mM
1M
Creatine kinase*
0.6 mg ml1
Mix the first four ingredients and add water to 1 l *Add DTT and creatine kinase to the amount of buffer needed at the time of use
Binding assay buffer (for methionyl tRNA þ eIF2GTP binding) Component
Final concentration
Stock
Amount
HEPES–KOH, pH 7.6
25 mM
1M
25 ml
KOAc, pH 7.6
80 mM
1M
80 ml
MgOAc
2.5 mM
1M
2.5 ml
DTT*
2 mM
1M
Mix the first three ingredients and add water to 1 l *Add DTT to the amount of buffer needed at the time of use
4. PROTOCOL 4.1. Preparation Prepare radiolabeled nucleic acid by the method of your choice. Prepare protein dilution buffer and binding assay buffer.
200
Sarah Kolitz and Jon R. Lorsch
Soak the filters in binding assay buffer for 1 h before beginning the protocol. Place additional binding assay buffer on ice for use in washing filters.
4.2. Duration Preparation
About 1 day
Protocol
About 2 h
4.3. Tip The protein dilution buffer and binding assay buffer presented here are for eukaryotic initiation factor 2 (eIF2) binding to eukaryotic initiator methionyl tRNA. The buffers used for other proteins may differ, but the protein dilution buffer will likely contain buffer to maintain pH, salt, glycerol, and DTT or other reducing agents. The addition of 0.6 mg ml1 creatine kinase will help prevent sticking of protein to tube walls; this can be an issue at low protein concentrations. Likewise, the binding assay buffer should be appropriate for the binding reaction of interest.
4.4. Caution Consult your institute Radiation Safety Officer for proper ordering, handling, and disposal of radioactive materials. See Fig. 16.1 for the flowchart of the complete protocol.
Figure 16.1 Flowchart of the complete protocol, including preparation.
Protein Filter Binding
201
5. STEP 1 ASSEMBLE BINDING REACTIONS 5.1. Overview Allow nucleic acid and protein to bind.
5.2. Duration 30 min 1.1 Make serial dilutions of the protein in protein dilution buffer. Make the dilutions 5 higher than the protein concentrations you wish to use in the binding assay (e.g., to measure binding to 10 mM protein, make a 5 stock of 50 mM). 1.2 Aliquot 16 ml binding buffer into tubes representing each point. 1.3 Add 4 ml of the appropriate 5 protein dilution to each reaction. 1.4 Include a no-protein reaction (by adding 4 ml protein dilution buffer without protein) to obtain counts representing the background for the assay. 1.5 Add at least 1000 counts of the radiolabeled nucleic acid of interest and incubate for the appropriate length of time for the reaction to reach equilibrium.
5.3. Tip If you lack information about the concentration range that will be appropriate, try a wide range (e.g., nM to mM) and use those results to choose the appropriate range of points to use in subsequent experiments.
5.4. Tip Reactions should be at least 20 ml final volume.
5.5. Tip For each reaction, the number of counts (in the radiolabeled nucleic acid) will be divided between two filters such that it is proportional to the extent of binding. In order to ensure that enough counts are observed on each filter in the lower range of the binding curve, add at least 1000 total counts per reaction.
5.6. Tip The incubation may vary depending on the time required for the binding reaction to reach equilibrium. This must be determined empirically.
202
Sarah Kolitz and Jon R. Lorsch
Figure 16.2 Flowchart of Step 1.
5.7. Tip In the example presented here for the binding of eukaryotic initiator methionyl tRNA to eIF2GTP, incubation for 10 min at 26 C is sufficient. If you are not sure of the kinetics of the interaction of interest, you may wish to try several incubation time points to make sure that the reaction has reached equilibrium (i.e., increase the incubation time and make sure that the fraction bound remains the same). See Fig. 16.2 for the flowchart of Step 1.
6. STEP 2 QUANTIFY BINDING 6.1. Overview Filter reactions and quantify the amount of radioactivity present on each filter.
6.2. Duration 30 min 2.1 With the vacuum off, place the Nytran SPC filter on the vacuum manifold, and then place the nitrocellulose filter on top of it. 2.2 Turn on the vacuum immediately before loading the sample in order to prevent the filters from drying out. Apply 20 ml of sample to the center of the filter. 2.3 Wash with 3 ml of cold binding assay buffer, taking care to wash the entire filter.
Protein Filter Binding
203
2.4 Allow the filters to dry for 1 min on the vacuum manifold, and then turn off the vacuum and carefully remove each filter to a separate, labeled scintillation vial. 2.5 Add 5 ml of scintillation fluid to each vial and count for the appropriate radioisotope using a scintillation counter.
6.3. Tip Make sure that each filter is intact and covers the entire surface to which the sample will be applied.
6.4. Tip It is essential to avoid switching the order of the filters!
6.5. Tip Alternatively, if the signal is sufficient, filters can be exposed to a phosphor screen and the signal can be quantified using ImageQuant or a similar software program. See Fig. 16.3 for the flowchart of Step 2.
Figure 16.3 Flowchart of Step 2.
204
Sarah Kolitz and Jon R. Lorsch
7. STEP 3 PROCESS BINDING DATA 7.1. Overview Obtain binding curves from the data.
7.2. Duration 15 min 3.1 For each point, add the counts from the top and bottom filters to obtain the total amount of radiolabeled nucleic acid present in the sample. 3.2 Divide the counts from the top filter, representing the amount of labeled nucleic acid bound to the protein of interest, by the total number of counts to obtain the fraction of labeled nucleic acid bound. Correct this value for background by subtracting the fraction obtained from the no protein reaction. 3.3 Using graphing software, such as Kaleidagraph or Excel, plot the fraction bound values versus protein concentration (Fig. 16.4). Fit with a quadratic (in the case of tight binding) or hyperbolic (in the case of
Figure 16.4 Data obtained from a filter binding assay monitoring [35 S]methionine-labeled initiator tRNA binding to eukaryotic initiation factor (eIF) 2. The data have been fit with a quadratic binding curve.
Protein Filter Binding
205
Figure 16.5 Flowchart of Step 3.
weak binding) binding isotherm to obtain an apparent Kd value for the interaction. See Fig. 16.5 for the flowchart of Step 3.
REFERENCES Referenced Literature Nirenberg, M., & Leder, P. (1964). RNA codewords and protein synthesis. Science, 145, 1399–1407. Oehler, S., Alex, R., & Barker, A. (1999). Is nitrocellulose filter binding really a universal assay for protein-DNA interactions? Analytical Biochemistry, 268, 330–336. Wong, I., & Lohman, T. M. (1993). A double filter method for nitrocellulose-filter binding: Application to protein-nucleic acid interactions. Proceedings of the National Academy of Sciences of the United States of America, 90, 5428–5432.
SOURCE REFERENCES Acker, M. G., Kolitz, S. E., Mitchell, S. F., Nanda, J. S., & Lorsch, J. R. (2007). Reconstitution of yeast translation initiation. Methods in Enzymology, 430, 111–145.
Protein Filter Binding Sarah Kolitz*,1, Jon R. Lorsch†
*Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA † Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 4.3 Tip 4.4 Caution 5. Step 1 Assemble Binding Reactions 5.1 Overview 5.2 Duration 5.3 Tip 5.4 Tip 5.5 Tip 5.6 Tip 5.7 Tip 6. Step 2 Quantify Binding 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 6.5 Tip 7. Step 3 Process Binding Data 7.1 Overview 7.2 Duration References Source References
198 198 198 199 199 199 200 200 200 201 201 201 201 201 201 201 202 202 202 202 203 203 203 204 204 204 205 205
Abstract This protocol describes a method to monitor the binding of nucleic acid to protein, allowing the determination of the apparent affinity of a nucleic acid–protein interaction. Methods in Enzymology, Volume 541 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420119-4.00016-1
#
2014 Elsevier Inc. All rights reserved.
197
198
Sarah Kolitz and Jon R. Lorsch
1. THEORY This method relies on the fact that most proteins will bind to a nitrocellulose filter, while free nucleic acid passes through. Thus if a radiolabeled nucleic acid is bound to a protein, it can be detected on the filter, whereas unbound nucleic acid will pass through (Nirenberg and Leder, 1964). In this way, a sequence of points at different protein concentrations or times can be observed in order to obtain thermodynamic binding curves or kinetic time courses for the interaction between the protein and nucleic acid. Since each concentration (or time) point is done using a separate filter, variability due to pipetting errors in the total amount of material added to each filter can contribute to noise in the data. This can be addressed by adding a filter that binds nucleic acid (such as Nytran™ SuPerCharge (Whatman)) below the nitrocellulose filter, such that variability in the amount of labeled nucleic acid loaded onto the filter can be taken into account (Wong and Lohman, 1993). Advantages of this technique include its relative ease and speed as well as the fact that it does not require particularly expensive equipment. Some proteins, however, bind poorly to nitrocellulose, or may be unable to bind nitrocellulose and nucleic acid simultaneously (Oehler et al., 1999); thus in certain situations this technique cannot be applied. In addition, if the rate of dissociation of the complex is high, it may fall apart during the washing phase of the protocol. In these cases, alternative approaches to monitoring binding must be undertaken, including native gel electrophoresis and fluorescence anisotropy.
2. EQUIPMENT Vacuum manifold Scintillation counter Micropipettors (P1000 and P20) Micropipettor tips 1.5-ml microcentrifuge tubes Whatman GF/C filters (24 mm) Nytran™ SuPerCharge (SPC) membranes
3. MATERIALS Radiolabeled nucleic acid of interest Protein of interest
199
Protein Filter Binding
HEPES Potassium hydroxide (KOH) Magnesium acetate (MgOAc) Potassium acetate (KOAc) Glacial acetic acid Glycerol Dithiothreitol (DTT) Creatine kinase Scintillation fluid
3.1. Solutions & buffers Step 1 Protein dilution buffer (for eIF2) Component
Final concentration
Stock
Amount
HEPES–KOH, pH 7.6
20 mM
1M
20 ml
KOAc, pH 7.6
100 mM
1M
100 ml
MgOAc
0.1 mM
1M
0.1 ml
Glycerol
10%
100%
100 ml
DTT*
2 mM
1M
Creatine kinase*
0.6 mg ml1
Mix the first four ingredients and add water to 1 l *Add DTT and creatine kinase to the amount of buffer needed at the time of use
Binding assay buffer (for methionyl tRNA þ eIF2GTP binding) Component
Final concentration
Stock
Amount
HEPES–KOH, pH 7.6
25 mM
1M
25 ml
KOAc, pH 7.6
80 mM
1M
80 ml
MgOAc
2.5 mM
1M
2.5 ml
DTT*
2 mM
1M
Mix the first three ingredients and add water to 1 l *Add DTT to the amount of buffer needed at the time of use
4. PROTOCOL 4.1. Preparation Prepare radiolabeled nucleic acid by the method of your choice. Prepare protein dilution buffer and binding assay buffer.
200
Sarah Kolitz and Jon R. Lorsch
Soak the filters in binding assay buffer for 1 h before beginning the protocol. Place additional binding assay buffer on ice for use in washing filters.
4.2. Duration Preparation
About 1 day
Protocol
About 2 h
4.3. Tip The protein dilution buffer and binding assay buffer presented here are for eukaryotic initiation factor 2 (eIF2) binding to eukaryotic initiator methionyl tRNA. The buffers used for other proteins may differ, but the protein dilution buffer will likely contain buffer to maintain pH, salt, glycerol, and DTT or other reducing agents. The addition of 0.6 mg ml1 creatine kinase will help prevent sticking of protein to tube walls; this can be an issue at low protein concentrations. Likewise, the binding assay buffer should be appropriate for the binding reaction of interest.
4.4. Caution Consult your institute Radiation Safety Officer for proper ordering, handling, and disposal of radioactive materials. See Fig. 16.1 for the flowchart of the complete protocol.
Figure 16.1 Flowchart of the complete protocol, including preparation.
Protein Filter Binding
201
5. STEP 1 ASSEMBLE BINDING REACTIONS 5.1. Overview Allow nucleic acid and protein to bind.
5.2. Duration 30 min 1.1 Make serial dilutions of the protein in protein dilution buffer. Make the dilutions 5 higher than the protein concentrations you wish to use in the binding assay (e.g., to measure binding to 10 mM protein, make a 5 stock of 50 mM). 1.2 Aliquot 16 ml binding buffer into tubes representing each point. 1.3 Add 4 ml of the appropriate 5 protein dilution to each reaction. 1.4 Include a no-protein reaction (by adding 4 ml protein dilution buffer without protein) to obtain counts representing the background for the assay. 1.5 Add at least 1000 counts of the radiolabeled nucleic acid of interest and incubate for the appropriate length of time for the reaction to reach equilibrium.
5.3. Tip If you lack information about the concentration range that will be appropriate, try a wide range (e.g., nM to mM) and use those results to choose the appropriate range of points to use in subsequent experiments.
5.4. Tip Reactions should be at least 20 ml final volume.
5.5. Tip For each reaction, the number of counts (in the radiolabeled nucleic acid) will be divided between two filters such that it is proportional to the extent of binding. In order to ensure that enough counts are observed on each filter in the lower range of the binding curve, add at least 1000 total counts per reaction.
5.6. Tip The incubation may vary depending on the time required for the binding reaction to reach equilibrium. This must be determined empirically.
202
Sarah Kolitz and Jon R. Lorsch
Figure 16.2 Flowchart of Step 1.
5.7. Tip In the example presented here for the binding of eukaryotic initiator methionyl tRNA to eIF2GTP, incubation for 10 min at 26 C is sufficient. If you are not sure of the kinetics of the interaction of interest, you may wish to try several incubation time points to make sure that the reaction has reached equilibrium (i.e., increase the incubation time and make sure that the fraction bound remains the same). See Fig. 16.2 for the flowchart of Step 1.
6. STEP 2 QUANTIFY BINDING 6.1. Overview Filter reactions and quantify the amount of radioactivity present on each filter.
6.2. Duration 30 min 2.1 With the vacuum off, place the Nytran SPC filter on the vacuum manifold, and then place the nitrocellulose filter on top of it. 2.2 Turn on the vacuum immediately before loading the sample in order to prevent the filters from drying out. Apply 20 ml of sample to the center of the filter. 2.3 Wash with 3 ml of cold binding assay buffer, taking care to wash the entire filter.
Protein Filter Binding
203
2.4 Allow the filters to dry for 1 min on the vacuum manifold, and then turn off the vacuum and carefully remove each filter to a separate, labeled scintillation vial. 2.5 Add 5 ml of scintillation fluid to each vial and count for the appropriate radioisotope using a scintillation counter.
6.3. Tip Make sure that each filter is intact and covers the entire surface to which the sample will be applied.
6.4. Tip It is essential to avoid switching the order of the filters!
6.5. Tip Alternatively, if the signal is sufficient, filters can be exposed to a phosphor screen and the signal can be quantified using ImageQuant or a similar software program. See Fig. 16.3 for the flowchart of Step 2.
Figure 16.3 Flowchart of Step 2.
204
Sarah Kolitz and Jon R. Lorsch
7. STEP 3 PROCESS BINDING DATA 7.1. Overview Obtain binding curves from the data.
7.2. Duration 15 min 3.1 For each point, add the counts from the top and bottom filters to obtain the total amount of radiolabeled nucleic acid present in the sample. 3.2 Divide the counts from the top filter, representing the amount of labeled nucleic acid bound to the protein of interest, by the total number of counts to obtain the fraction of labeled nucleic acid bound. Correct this value for background by subtracting the fraction obtained from the no protein reaction. 3.3 Using graphing software, such as Kaleidagraph or Excel, plot the fraction bound values versus protein concentration (Fig. 16.4). Fit with a quadratic (in the case of tight binding) or hyperbolic (in the case of
Figure 16.4 Data obtained from a filter binding assay monitoring [35 S]methionine-labeled initiator tRNA binding to eukaryotic initiation factor (eIF) 2. The data have been fit with a quadratic binding curve.
Protein Filter Binding
205
Figure 16.5 Flowchart of Step 3.
weak binding) binding isotherm to obtain an apparent Kd value for the interaction. See Fig. 16.5 for the flowchart of Step 3.
REFERENCES Referenced Literature Nirenberg, M., & Leder, P. (1964). RNA codewords and protein synthesis. Science, 145, 1399–1407. Oehler, S., Alex, R., & Barker, A. (1999). Is nitrocellulose filter binding really a universal assay for protein-DNA interactions? Analytical Biochemistry, 268, 330–336. Wong, I., & Lohman, T. M. (1993). A double filter method for nitrocellulose-filter binding: Application to protein-nucleic acid interactions. Proceedings of the National Academy of Sciences of the United States of America, 90, 5428–5432.
SOURCE REFERENCES Acker, M. G., Kolitz, S. E., Mitchell, S. F., Nanda, J. S., & Lorsch, J. R. (2007). Reconstitution of yeast translation initiation. Methods in Enzymology, 430, 111–145.
