| Name: | Mouse monoclonal antibody to Nop1p |
| Immunogen: | Yeast nuclear preparation |
| HGNC Name: | FBL |
| UniProt: | P15646 |
| Molecular Weight: | 34.5kDa |
| Host: | Mouse |
| Isotype: | IgG3 |
| Species Cross-Reactivity: | Saccharomyces cerevisiae |
| RRID: | AB_2572368 |
| Format: | Concentrated hybridoma cell culture media plus 5mM NaN3 |
| Applications: | WB, ICC/IF, IP |
| Recommended Dilutions: | WB: 1:1,000. ICC/IF: 1:2,000. |
| Storage: | Store at 4°C for short term, for longer term at -20°C. Avoid freeze/thaw cycles. |
Western blots of whole yeast protein extracts with a collection of our antibodies. The blot for MCA-28F2 is in the indicated lane, and the number indicates the SDS-PAGE molecular weight in kiloDaltons.
Mouse Monoclonal Antibody to Nop1p
Cat# MCA-28F2
$120.00 – $800.00
Nop1p was originally identified as a nucleolar protein of the bakers yeast Saccharomyces cerevisiae (1). It was later found to be highly conserved in protein sequence and the mammalian homologue is fibrillarin, the two protein being 67% identical (2,3). Fibrillarin is a highly conserved component of a nucleolar small ribonucleoprotein complex in mammals, involved in the processing of ribosomal RNA during ribosomal biogenesis. Both proteins runs at ~35kDa on SDS-PAGE and is very rich in basic amino acids having a PI of 9.8. An alternate name for the protein is “U3 small nuclear ribonucleoprotein” (U3snRNP). The Nop1p/fibrillarin molecule consists of an N-terminal glycine and arginine rich region followed by a highly conserved globular domain. Embryonic knockout of the fibrillarin gene in mice is lethal, suggesting fundamental importance of this protein (4). Autoantibodies to fibrillarin are also seen in patients with the autoimmune disease systemic sclerocis (5).
MCA-28F2 does not recognize the mammalian fibrillarin, although we have several mouse, rabbit and chicken antibodies which do. From the same series of antibodies another clone binds both Nop1p and human fibrillarin, MCA-38F3. This has become widely used as a marker of nucleoli. Other EnCor antibodies were made against recombinant human fibrillarin, rabbit and chicken polyclonal antibodies RPCA-Fib and CPCA-Fib. An alternate mouse monoclonal antibody MCA-4A4 was also made against recombinant human fibrillarin.
Nop1p was originally identified as a nucleolar protein of bakers yeast, Saccharomyces cerevisiae(accession P15646). The Nop1p protein is essential for yeast viability and is localized in the nucleoli. The human homologue of Nop1p is fibrillarin (accession P22087) a component of the nucleolar small nuclear ribonucleoprotein (snRNP) particle. The human fibrillarin gene is located on chromosome 19 (19q13.1). Fibrillarin proteins have been cloned and sequenced from several other species (Mouse, accession P35550, Xenopus accession P22232, C. elegans accession Q22053, and S. pombe accession P35551. You can download a pdf of the sequence alignment here.
The N terminal ~80 amino acids contain multiple copies based on the peptide RGG, and the remaining ~240 amino acids consist of the fibrillarin domain. A fibrillarin homologue has also been identified in the genome of the archean Methanococcus (accession NC_000909). This protein lacks the RGG rich N-terminal extension but is clearly homologues to the other sequences throughout all of the fibrillarin domain. The structure of this molecule has been determined and shown to consist of 2 extended b-sheets flanked by 4 a-helixes (Medline link).
Patients with the autoimmune disease scleroderma often have circulating autoantibodies to a 34 kDa protein which was subsequently found to be fibrillarin.
To raise this antibody, mice were injected with yeast nuclear preparations and hybridomas were screened by immunofluorescence on yeast cells and by western blotting on yeast protein homogenates. This monoclonal antibody does not cross-react with fibrillarin in other species, but is an excellent marker of the nucleolus of yeast cells. For Nop1p/fibrillarin visualization in vertebrates, Drosophila and C. elegans and other species we recommend MCA-38F3, also available from EnCor.
Antibody is supplied as a concentrated tissue culture supernatant, sterile-filtered, from an Integra CL-350 biochamber plus sodium azide. The immunoglobulin subtype is IgG3. The concentration of IgG is unknown. Store at 4°C or -20°C. For western blots of yeast protein samples, use MCA-28F2 diluted 1/10,000 (cell lysates) to 1/25,000 (nuclear fractions), followed by chemiluminescent detection (ECL). See here for image of blot. For other (non-ECL) western detection methods, try MCA-28F2 diluted 1/1000 to 1/5000. For immunofluorescence on yeast cells, use MCA-28F2 diluted 1/2000 to 1/20,000. We routinely dilute to 1/5000. For immunoprecipitations, use 2-4 µl for an amount of lysate derived from 5 OD600 units of yeast culture.
1. Aris JP and Blobel G. Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J. Cell Biol. 107:17-31 (1988).
2. Aris JP and Blobel G. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera. Proc. Natl. Acad. Sci. 88:931-5 (1991).
3. Ochs RL, Lischwe MA, Spohn WH, Busch H. Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. Biol. Cell. 54:123-33 (1985).
4. Newton K, Petfalski E, Tollervey D, Caceres JF. Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse. Mol. Cell Biol. 23:8519-27 (2003).
5. Okano Y, Steen VD, Medsger TA. Autoantibody to U3 nucleolar ribonucleoprotein (fibrillarin) in patients with systemic sclerosis. Arth. Rheum. 35:95-100 (1992).
