408

NOTES

sized with a primer complementary to a sequence near the 5’ end of the CK or Cr region (Table l), with the cDNA synthesis system of Promega, Inc. (Madison, WI). cDNA was cloned into the plasmid vector pGEM3Zf(-) (Promega). PCR amplification was then performed with an upstream primer from the vector and a downstream primer complementary to a sequence in the CT or CK region (but just upstream from the primer used for initial cDNA synthesis) (Table 1). Amplified cDNA was cloned into plasmid vector pCRlOO0 and transformed into INVaF’ competent cells using a TA cloning system (Invitrogen, San Diego, CA). Without further selection of the transformed colonies, we found that not all of them contained Ig cDNA (Fig. la), and we obtained two different VL cDNA clones from each hybridoma, as have others (9). One of the cDNAs is derived from the B cell. The second is an aberrant, nonproductive mRNA transcript of the SP2 fusion partner (9). In the SP2 VL, four nucleotides were deleted at the VKJK junction, resulting in a frameshift and premature termination codon after the J sequence. The same is true for other frequently used fusion partners derived from the original MOPC-21 tumor (9). (Myelomas P3-X63Ag8, X63-Ag8.653, and NSl are cells that originate from MOPC-21.) SP2 cells do not synthesize H chain mRNA, and we consistently obtained a single species of H chain VDJ cDNA from each hybridoma. Selection of colonies for sequencing was more efficient when we screened colonies to identify the B cell VL cDNA clones. After transformation of bacteria with &-amplified cDNA, white colonies were dotted in a grid array onto a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The probe for a first hybridization on this membrane was an oligonucleotide complementary to the CK region, upstream from the primer used for amplification (Table 1). This probe hybridized with 430 of 500 white cDNA clones from hybridoma 2ClO (Fig. 1) and 50 of 110 white clones from H241 (not shown). The membrane was washed with boiling water, removal of all radioactivity was confirmed by film exposure, and a second hybridization was then performed with SPB-specific oligonucleotide corresponding to the VKJK region (Table 1). The SP2-specific probe hybridized with 100 of the 430 &-positive clones of 2ClO (Fig. 1) and with 3 of the 50 white clones from H241. Thus, relative expression of the SP2 and B cell L chains is highly variable in different hybridomas. On sequencing clones that were positive with the CK probe and negative with the SP2-specific CDR3 probe, we found only the fully translatable sequence of the hybridoma product. Thus, the SP2 VKJK junction oligonucleotide is a useful probe for the identification of B cell VL cDNA clones from hybridomas made with SP2 or related myelomas. This probe could also serve as a useful control in experiments meant to quantify hybridoma L chain mRNA production.

& TIPS REFERENCES 1. Kijhler, 2. KShler,

G., and Milstein, G., and Milstein,

3. Kiihler,

G., Howe,

C. (1975) C. (1976)

Nature 256, 495-497. Eur. J. Immunol. 6, 511-519.

S. C., and Milstein,

C. (1976)

Eur.

J. Immunol.

6,292-295. 4. Margulies, D. H., Kuehl, W. M., and Scharff, M. D. (1976) Cell 8, 405-415. 5. Schulman, M., Wilde, C. D., and K6hler, G. (1978) Nature 276, 269-270. 6. Kearney, J. F., Radbrush, A., Liesegang, B., and Rajewsky, K. (1979)J.Immunol. 123,1548-1550. 7. Stollar, B. D., Zon, G., and Pastor, R. W. (1986) hoc. N&l. Acad. Sci. USA 83,4469-4473. 8. Kubota,

T., Akatsuka,

T., and Kanai,

Y. (1986)

Zmmunol.

Lett.

14,53-58. 9. Carroll, 991-995.

W. L., Mendel,

Custom-Made for Low-Speed

E., and Levy,

S. (1988)

Mol.

Adapters in Silicone Centrifugation

Immunol.

25,

Rubber

Victor W. T. Wong of Zoology, Singapore

Department Kent Ridge,

National 0511

University

of Singapore,

Many commercial makes of plastic tubes may be adapted for centrifugation if a suitable adapter can be found. Silicone rubber was used to make custom-made adapters for low-speed centrifugation. Most labs are fitted with benchtop centrifuges of about 6000 rpm maximal capacity and a bewildering array of centrifuge tubes from various manufacturers,

CENTRIFUGE

V-SHAPED TRANSPARENCY

CENTRIFUGE

TUBES

STRUTS FI

BUCKET

FIG. 1.

Setup for making silicone rubber adapters. The centrifuge tubes are kept upright and aligned by a glass plate. A weight (omitted for clarity) is placed on top of this glass plate. ANALYTICAL

All

BIOCHEMISTRY Copyright 0 1992 rights of reproduction

204,408-409 (1992) 0003-2697192 55.00 by Academic Press, Inc. in any form reserved.

NOTES

which, if suitable for centrifugation, are not suitable for other biochemical manipulations. This necessitates switching to centrifuge tubes for the centrifugation steps and back again, involving unnecessary delay. Many commercial makes of tubes may be adapted for centrifugation if a suitable adapter can be found. This paper describes a quick and economical method for making custom-made adapters which are suitable for the numerous low-speed centrifugation tasks required for biochemical techniques. The adapters were made for Materials and methods. a colleague who required sucrose step gradients to be set up in numerous plastic centrifuge tubes. These tubes were of an unknown make but were required to fit into a Hettich universal benchtop centrifuge (Hettich Zentrifugen, D-7200 Tuttlingen, Germany) fitt.ed with four swing-out buckets. The material used was silicone rubber 3120RTV red (Dow Corning Corp., Midland, MI). This comes supplied in a l-lb tin together with a catalyst (S standard curve). The insides of the swing-out buckets were covered with a rolled layer of transparent acetate film to allow some clearance. To add air channels, V-shaped struts may be lightly tacked into place onto the transparent film. Uhu adhesive or Cow gum is suitable for these temporary joints. The silicone rubber was thoroughly mixed with the catalyst at a ratio of lO:l, using a wooden spatula, and poured into the buckets to a predetermined height, allowing for displacement of some of the silicone rubber by the cent,rifuge tubes. Because numerous centrifuge tubes were required, it was decided to fit three tubes into one bucket. These tubes were taped together and inserted into the silicone rubber. The buckets were placed close to each other. A glass plate with a weight was placed on top of the centrifuge tubes to align and keep

FIG.

2.

Photograph

of silicone

rubber

adapters.

409

& TIPS

them upright (Fig. 1). The mixture was left to set overnight. The molds were removed the Results and discussion. next day by gripping the transparency film and struts with a pair of pliers. The molds were lightly dusted with talcum powder to remove any tackiness in the silicone material. They were then balanced against each other by trimming and pairing excess silicone rubber from the top (Fig. 2). These custom-made adapters are easy to make and very convenient to use. They are capable of withstanding the low-speed centrifugation tasks for which they were constructed. Achnourledgment. graph.

I thank

Shawn

K. Y. Lum

for taking

the photo-

A General Method for the Detection and Quantitation of Antigens in Solubilized Cells Using Radiolabeled Fab Fragment Joe B. Harford Cell Biology & Metabolism Branch, National Institute Health & Human Development, National Institutes of Health, Bethesda, Maryland 20892

of Child

A number of cellular receptors have been assayed with radiolabeled ligand after solubilization of cells in nonionic detergents. These “soluble receptor assays” are dependent on the retention of ligand-binding activity by the solubilized receptor and the ability to separate receptor-bound ligand from a relatively large amount of unbound ligand. One common method for such separation involves precipitation of the receptor-ligand complex with agents like ammonium sulfate (l-3) or polyethylene glycol (4-7) under conditions which do not precipitate free radiolabeled ligand. As the ligand in question becomes larger (e.g., >50,000 Da), it becomes progressively more difficult to precipitate selectively the receptor-ligand complex without significant precipitation of unbound ligand. As a result, the signal-tonoise ratio for a soluble receptor assay can become prohibitively low with larger ligands. In addition to this restriction on the size of the ligand, these methods are not applicable to cellular proteins which are not receptors. Here, a method that is based on the principles of a soluble receptor assay but which utilizes radiolabeled Fab fragment in place of ligand is presented. The method has been used to assay endogenous transferrin receptors (TfR) in a human cell line and to detect human TfR following transfection of an expression vector into murine cells. While the method’s utility is demonstrated for the human TfR, it could also be used for ANALYTICAL

BIOCHEMISTRY

Copyright 0 1992 All rights of reproduction

204,

409-411

(1992)

0003.x97/92 $5.00 by Academic Press, Inc. in any form reserved.

Custom-made adapters in silicone rubber for low-speed centrifugation.

408 NOTES sized with a primer complementary to a sequence near the 5’ end of the CK or Cr region (Table l), with the cDNA synthesis system of Promeg...
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