EnCor Biotechnology
Mouse Monoclonal Antibody to Nsp1p Cat# MCA-32D6
Description
The MCA-32D6 antibody was originally made against a fraction from a yeast nuclear preparation which was rich in nucleoli (2). The antibody recognized yeast nuclear pore complexes in immunofluorescence experiments. Subsequently a screen of a λgt11 expression library yielded a single positive clone carrying an insert encoding ~66% of the C-terminal portion of Nsp1p, and subsequent western blotting of yeast extracts was consistent with this (2,3). This antibody is therefore a useful marker of yeast nuclear pores.
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| Name: | Nsp1p, mouse monoclonal antibody, Cat# MCA-32D6 |
| Immunogen: | Yeast nuclear preparations |
| HGNC Name: | N.A. |
| UniProt: | G2WGX7 |
| Molecular Weight: | 86.5kDa |
| Host: | Mouse |
| Isotype: | IgG1 |
| Species Cross-Reactivity: | Sacchoromyces cerevisiae |
| RRID: | AB_2157646 |
| Format: | Concentrated hybridoma tissue culture media plus 5mM NaN3 |
| Applications: | WB, IF/ICC, IHC, IP |
| Recommended Dilutions: | WB: 1:10,000 (cell lysates), 1:50,000 (nuclear fractions). IF/ICC and IHC: 1:1,000-1:10,000. |
| Storage: | Store at 4°C for short term, for longer term at -20°C. Avoid freeze/thaw cycles. |
MCA-32D6 recognizes Nsp1p, a nuclear pore complex protein, and this antibody is a useful marker of yeast nuclear pores. The monoclonal antibody MCA-32D6 was initially identified as directed against a nuclear pore complex antigen by immunofluorescence localization. A screen of a lgt11 expression library yielded a single positive clone carrying an insert encoding ~66% of the C-terminal portion of Nsp1p. To confirm that MCA-32D6 possessed high affinity for Nsp1p, strain RS453, which expresses a shortened isoform of Nsp1p, was compared to the wildtype strain BJ5465. NSP1 in the RS453 strain contains an internal deletion that removes the coding sequence for 6 FXFG repeats, which are not essential for protein function, and encodes a protein that has been observed to migrate at approximately 85 kDa on SDS-PAGE gels. The predicted size of wildtype Nsp1p is 86.5 kDa, but Nsp1p has been observed to migrate on SDS gels at ~100 kDa. In our SDS-PAGE gels, the apparent molecular mass of wild type Nsp1p was 108 kDa, whereas the isoform of Nsp1p expressed in the RS453 strain migrated at 91 kDa. The detection of two protein bands of apparent sizes 108 kDa and 91 kDa in the BJ5465 and RS453 strains, respectively, demonstrated that MCA-32D6 recognized the pore complex protein Nsp1p.
For western blots of yeast protein samples, use MCA-32D6 diluted 1/10,000 (cell lysates) to 1/50,000 (nuclear fractions), followed by chemiluminescent detection (ECL). Press here for image of blot. For other (non-ECL) western detection methods, try MCA-32D6 diluted 1/1,000 to 1/5,000. For immunofluorescence on yeast cells, use MCA-32D6 diluted 1/1,000 to 1/10,000.
Comprehensive Yeast Genome Database (CYGD) Link: press here.
Saccharomyces Genome Database (SGD) Link: press here.
This antibody to a yeast protein is not appropriate for use on mammalian tissues for western blotting, IF, ICC or IHC.
1. Belgareh N, et al. Functional characterization of a Nup159p-containing nuclear pore subcomplex. Mol. Biol. Cell 9:3475-92 (198)..
2. Tolerico LH, et al. Saccharomyces cerevisiae Mod5p-II contains sequences antagonistic for nuclear and cytosolic locations. Genetics 151:57-75 (1999).
3. Fahrenkrog B, et al. Comparative spatial localization of protein-A-tagged and authentic yeast nuclear pore complex proteins by immunogold electron microscopy. J. Struct. Biol. 129:295-305 (2000).
Peer Reviewed Articles using this antibody
1. Brickner DG, Light W, Brickner JH. Quantitative localization of chromosomal loci by immunofluorescence. Methods Enzymol. 470:569-80 (2010).
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