Immunogen:  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 kDal on SDS-PAGE gels. The predicted size of wildtype Nsp1p is 86.5 kDal, but Nsp1p has been observed to migrate on SDS gels at ~100 kDal. In our SDS-PAGE gels, the apparent molecular mass of wild type Nsp1p was 108 kDal, whereas the isoform of Nsp1p expressed in the RS453 strain migrated at 91 kDal. The detection of two protein bands of apparent sizes 108 kDal and 91 kDal in the BJ5465 and RS453 strains, respectively, demonstrated that MCA-32D6 recognized the pore complex protein Nsp1p.

Diagram of Domain Structure: Generated from sequence of yeast Nsp1p with the SMART program from EMBL in Heidleberg. The pink regions are regions of low complexity, mostly rich in asparagine, glycine and serine. The block marked "PFAM Nsp1_C" corresponds to the Nsp1-like C-terminal region which is probably an a-helical coiled-coil region involved in heterodimerization with other molecules (Nup82p in the case of yeast). Scale is number of amino acids;

Antibody specificity (above): Western blots of whole yeast protein extracts with a collection of our antibodies. The blot for MCA-32D6 is in the indicated lane, and the number indicates the SDS-PAGE molecular weight in kiloDaltons.

Antibody Characteristics: The antibody is an IgG which stains the nuclear pore complex. It is supplied as a sterile-filtered cell culture fluid from an Integra CL-350 biochamber plus sodium azide. The immunoglobulin subtype and concentration are unknown.

Suggestions for use: 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. Antibody preparation contains 10mM sodium azide preservative (Press here for Material Safety Data Sheet). 4°C or -20°C. Avoid repeat freezing and thawing.

References:

Tolerico, L. H., A. L. Benko, J. P. Aris, D. R. Stanford, N. C. Martin, and A. K. Hopper. Saccharomyces cerevisiae Mod5p-II contains sequences antagonistic for nuclear and cytosolic locations. Genetics 151:57-75 (1999).

Fahrenkrog, B., J. P. Aris, E. C. Hurt, N. Pante, and U. Aebi. 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)

Availability and Price: Available for shipping now, $200 US per aliquot of 500ml of concentrated tissue culture derived material, enough for hundreds of experiments. For order form press here

Comprehensive Yeast Genome Database (CYGD) Link: press here.

Saccharomyces Genome Database (SGD) Link: press here.

Limitations: This product is for research use only and is not approved for use in humans or in clinical diagnosis.