Anti-Fibrillarin antibody - Nucleolar Marker (ab5821)

Overview

  • Product nameAnti-Fibrillarin antibody - Nucleolar MarkerSee all Fibrillarin primary antibodies ...
  • Description
    Rabbit polyclonal to Fibrillarin - Nucleolar Marker
  • SpecificityThis antibody detects a band at close to 34kDa in all species tested. The band can be completely blocked with the immuising peptide in all cases - this is very strong evidence that the antibody is recognising fibrillarin.
  • Tested applicationsIHC - Wholemount, ICC/IF, IP, IHC-P, WB, ICC more details
  • Species reactivity
    Reacts with: Mouse, Human, Fruit fly (Drosophila melanogaster)
    Predicted to work with: Xenopus laevis

    Does not react with

    Rat
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Fibrillarin.

    (Peptide available as ab13745.)

  • Positive control
    • This antibody gave a positive signal in the following whole cell lysates: HeLa; A431; Jurkat; 293T. This antibody also gave a positive signal in HeLa nuclear lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab5821 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
IHC - Wholemount Use at an assay dependent concentration. PubMed: 22291607
ICC/IF Use at an assay dependent dilution.
IP Use at an assay dependent dilution. PubMed: 19430468
IHC-P 1/100.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 39 kDa (predicted molecular weight: 34 kDa).Can be blocked with Human Fibrillarin peptide (ab13745).
ICC 1/100.

Target

  • FunctionInvolved in pre-rRNA processing. Utilizes the methyl donor S-adenosyl-L-methionine to catalyze the site-specific 2'-hydroxyl methylation of ribose moieties in pre-ribosomal RNA. Site specificity is provided by a guide RNA that base pairs with the substrate. Methylation occurs at a characteristic distance from the sequence involved in base pairing with the guide RNA.
  • Sequence similaritiesBelongs to the methyltransferase superfamily. Fibrillarin family.
  • Post-translational
    modifications
    By homology to other fibrillarins, some or all of the N-terminal domain arginines are modified to asymmetric dimethylarginine (DMA).
  • Cellular localizationNucleus > nucleolus. Fibrillar region of the nucleolus.
  • Target information above from: UniProt accession P22087 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links
    see all
  • Alternative names
    • 34 kD nucleolar scleroderma antigen antibody
    • 34 kDa nucleolar scleroderma antigen antibody
    • 34-kD nucleolar scleroderma antigen antibody
    • FBL antibody
    • FBRL_HUMAN antibody
    • FIB antibody
    • FIB1 antibody
    • FLRN antibody
    • Nop1p antibody
    • RNA U3 small nucleolar interacting protein 1 antibody
    • RNU3IP1 antibody
    • rRNA 2' O methyltransferase fibrillarin antibody
    • rRNA 2'-O-methyltransferase fibrillarin antibody
    see all

Anti-Fibrillarin antibody - Nucleolar Marker images

  • The cells were 100% methanol fixed (5 min) and then
    incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab37266, 1µg/ml, red) and (ab5821, 1µg/ml, blue) overnight at +4°C. The secondary antibodies were ab150115 Alexa Fluor® 647 goat anti-mouse IgG (H+L) used at 2µg/ml for 1h and ab175652 Alexa Fluor® 405 goat anti-rabbit IgG (H+L) used at 2µg/ml for 1h. 

  • ICC/IF image of ab5821 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab5821, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

  • Immunofluorescent imaging of human cells (U2OS) with ab5821 reveals highly specific localisation to the dense fibrillar component (DFC) of the nucleolus associated with the initial ribosomal RNA (rRNA) precursor. The nucleolar protein fibrillarin is located primarily in the DFC.  Blue is hoechst staining of the nucleus, green is ab5821 used at 1/100, merge image demonstrates exclusively nuclear localisation.  

    IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary antibody used at 1/100 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes.  All blocking and incubation steps carried out at 37 degrees C.

  • ab5821 staining human liver tissue sections by IHC-P.  Sections were formaldehyde fixed, permeabilized in Triton-X and subjected to heat mediated antigen retrieval prior to blocking with 6% BSA for 2 hours at 23°C. The primary antibody was diluted 1/100 and incubated with the sample for 16 hours at 5°C.  ab6717 was used as the secondary antibody.

    See Abreview

  • All lanes : Anti-Fibrillarin antibody - Nucleolar Marker (ab5821) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 3 : Schneider L2 (Fruit fly (Drosophila melanogaster)) Whole Cell Lysate (ab14893)

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size : 34 kDa
    Observed band size : 37 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 42 kDa,70 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 3 minutes
  • Immunohistochemical analysis of salivary glands from Drosophila melanogaster larvae, staining Fibrillarin with ab5821. Tissues were fixed in 4% paraformaldehyde/PBS and 0.3% Triton-X/PBS before incubation with primary antibody.

References for Anti-Fibrillarin antibody - Nucleolar Marker (ab5821)

This product has been referenced in:
  • Tessarz P  et al. Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification. Nature 505:564-8 (2014). WB . Read more (PubMed: 24352239) »
  • Schenkwein D  et al. rDNA-directed integration by an HIV-1 integrase--I-PpoI fusion protein. Nucleic Acids Res 41:e61 (2013). ICC/IF ; Human . Read more (PubMed: 23275537) »

See all 35 Publications for this product

Product Wall

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (MRC-5)
Specification MRC-5
Fixative Paraformaldehyde
Permeabilization Yes - 0.2% Triton X-100
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: RT°C
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Submitted Feb 11 2013

Thank you for your response. Unfortunately, we do not have the pdf format of our on-line product datasheets yet but IT Department will consider implementing it. I have saved the on-line documents as word documents and attached the files ...

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Thank you for your enquiry and your interest. The datasheets of these products can be found at our website: http://www.abcam.com/fibrillarin-antibody-nucleolar-marker-ab5821.html http://www.abcam.com/nop5p-nop56p-antibody-37c12-ab...

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As requested, I have issued a free of charge replacement, ab4566, mouse fibrillarin antibody 38F3. To check the status of the order please contact our Customer Service team and reference this number. You should expect the antibody on Tuesday of next...

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Application Western blot
Sample Human Cell lysate - nuclear (Non-small cell lung carcinoma)
Loading amount 10 µg
Specification Non-small cell lung carcinoma
Gel Running Conditions Reduced Denaturing (4-12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Dec 01 2010

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cultured Cells (HeLa)
Specification HeLa
Fixative Paraformaldehyde
Permeabilization Yes - .25% triton x-100 10min RT
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 24°C
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Submitted Nov 29 2010

Application Western blot
Sample Human Cell lysate - whole cell (HeLa)
Loading amount 20 µg
Specification HeLa
Gel Running Conditions Reduced Denaturing (10% gel)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
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Submitted Nov 29 2010

Application Western blot
Sample Human Cell lysate - other (HeLa cells)
Loading amount 20 µg
Specification HeLa cells
Gel Running Conditions Reduced Denaturing (PAGE 12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
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Mr. CESAR CAMACHO

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Submitted May 12 2010

Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (Primary neuronal culture)
Specification Primary neuronal culture
Fixative Paraformaldehyde
Permeabilization Yes - 0.3% Triton X-100
Blocking step Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
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Submitted Feb 11 2010

Application Immunocytochemistry
Sample Human Cultured Cells (a7 cells, human melanoma cell line)
Specification a7 cells, human melanoma cell line
Fixative Paraformaldehyde
Permeabilization Yes - triton 0.1%
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: rt°C
Username

Mr. CESAR CAMACHO

Verified customer

Submitted Jun 24 2009

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"