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2159
Fibrillarin (R122) Antibody (IF Specific)
Primary Antibodies

Fibrillarin (R122) Antibody (IF Specific) #2159

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# Product Name Application Reactivity
  • WB
  • IF
H M R Mk
Filter:
  1. IF

Confocal immunofluorescent analysis of COS cells using Fibrillarin (R122) Antibody (IF Specific) (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Supporting Data

REACTIVITY Mk
SENSITIVITY Endogenous
MW (kDa) 37
SOURCE Rabbit

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Immunofluorescence (Immunocytochemistry) 1:25

Storage:

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.

Protocol

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

    NOTE: Formaldehyde is toxic, use only in a fume hood.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

Protocol Id: 24

Specificity / Sensitivity

Fibrillarin (R122) Antibody (IF Specific) detects endogenous levels of total fibrillarin protein by immunofluoresence (immunocytochemistry).

Species Reactivity:

Monkey

Species predicted to react based on 100% sequence homology:

Human, Mouse, Rat

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg122 of human fibrillarin. Antibodies are purified by protein A and peptide affinity chromatography.

Background

Fibrillarin is a 2'-O-methyltransferase located in fibrillar regions and Cajal bodies of the nucleolus, where RNA transcription and pre-RNA processing take place (1,2). Fibrillarin associates with several other structural proteins as well as box C/D snoRNA to form a complex that functions in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly. This complex catalyzes site-specific 2'-O-ribose methylation of targeted nucleotides within the rRNA sequence (3,4). The sequence, structure, and function of fibrillarin are highly conserved and fibrillarin gene expression is essential for early embryonic development (5).

  1. Tollervey, D. et al. (1993) Cell 72, 443-57.
  2. Wang, H. et al. (2000) EMBO J 19, 317-23.
  3. Lafontaine, D.L. and Tollervey, D. (2000) Mol Cell Biol 20, 2650-9.
  4. Galardi, S. et al. (2002) Mol Cell Biol 22, 6663-8.
  5. Newton, K. et al. (2003) Mol Cell Biol 23, 8519-27.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
DRAQ5 is a registered trademark of Biostatus Limited.

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