Overview

  • Product name
    Anti-hnRNP A1 antibody [9H10]
    See all hnRNP A1 primary antibodies
  • Description
    Mouse monoclonal [9H10] to hnRNP A1
  • Host species
    Mouse
  • Tested applications
    Suitable for: Flow Cyt, IHC-P, ELISA, WB, IP, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Full length hnRNPA1 native protein (partially purified) from HeLa cells (Human).

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.1% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity
    Protein A purified
  • Purification notes
    Purified from tissue culture supernatant.
  • Clonality
    Monoclonal
  • Clone number
    9H10
  • Myeloma
    Sp2/0
  • Isotype
    IgG2b
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab5832 in the following tested applications.

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

Application Abreviews Notes
Flow Cyt Use 1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ELISA Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Detects a band of approximately 34 kDa (predicted molecular weight: 38 kDa).
IP Use at an assay dependent concentration. This antibody does not IP the hnRNP complex.
ICC/IF Use at an assay dependent concentration. See Abreview (April 2, 2007)

Target

  • Function
    Involved in the packaging of pre-mRNA into hnRNP particles, transport of poly(A) mRNA from the nucleus to the cytoplasm and may modulate splice site selection. May play a role in HCV RNA replication.
  • Sequence similarities
    Contains 2 RRM (RNA recognition motif) domains.
  • Post-translational
    modifications
    Arg-194, Arg-206 and Arg-225 are dimethylated, probably to asymmetric dimethylarginine.
    Sumoylated.
  • Cellular localization
    Nucleus. Cytoplasm. Localized in cytoplasmic mRNP granules containing untranslated mRNAs. Shuttles continuously between the nucleus and the cytoplasm along with mRNA. Component of ribonucleosomes. In the course of viral infection, colocalizes with HCV NS5B at speckles in the cytoplasm in a HCV-replication dependent manner.
  • Information by UniProt
  • Database links
  • Alternative names
    • HNRNPA 1 antibody
    • Helix destabilizing protein antibody
    • Helix-destabilizing protein antibody
    • Heterogeneous nuclear ribonucleoprotein A1 antibody
    • Heterogeneous nuclear ribonucleoprotein A1B protein antibody
    • Heterogeneous nuclear ribonucleoprotein B2 protein antibody
    • Heterogeneous nuclear ribonucleoprotein core protein A1 antibody
    • hnRNP A1 antibody
    • hnRNP core protein A1 antibody
    • HNRNPA1 antibody
    • HNRPA1 antibody
    • MGC102835 antibody
    • Nuclear ribonucleoprotein particle A1 protein antibody
    • ROA1_HUMAN antibody
    • Single strand DNA binding protein UP1 antibody
    • Single strand RNA binding protein antibody
    • Single-strand RNA-binding protein antibody
    see all

Images

  • All lanes : Anti-hnRNP A1 antibody [9H10] (ab5832) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 38 kDa
    Observed band size: 37 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 43 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 30 seconds
  • ab5832 at 1/2000 staining GM10725 cells (Lymphoblastoid cell line). The cells were paraformaldehyde fixed and blocked with BSA prior to incubation with the antibody for 12 hours. An Alexa-Fluor ® 594 conjugated donkey anti-mouse antibody was used as the secondary. Cells were mounted in DAPI and observed using a confocal microscope.

    See Abreview

  • Ab5832 staining human lung. Staining is localized to the nucleus.
    Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
    Sections were stained using an automated system (Dako PT Link), at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer, citrate pH 6.0. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
  • All lanes : Anti-hnRNP A1 antibody [9H10] (ab5832) at 1/1000 dilution

    Lane 1 : Mouse NSC34 whole cell lysate with control siRNA
    Lane 2 : Mouse NSC34 whole cell lysate with hnRNP A1 siRNA

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : IRDye® 700DX-conjugated Donkey anti-Mouse IgG at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size: 38 kDa


    Exposure time: 1 minute


    Knockdown for 72 hours.

    See Abreview

  • Overlay histogram showing Jurkat cells stained with ab5832 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab5832, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.

  • hnRNP A1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to hnRNP A1 (ab5832) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab5832.
    Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
    Band: 37kDa: hnRNP A1; 42kDa:We are unsure as to the identity of this extra band.

References

This product has been referenced in:
  • Thompson MG  et al. Co-regulatory activity of hnRNP K and NS1-BP in influenza and human mRNA splicing. Nat Commun 9:2407 (2018). Read more (PubMed: 29921878) »
  • Hock EM  et al. Hypertonic Stress Causes Cytoplasmic Translocation of Neuronal, but Not Astrocytic, FUS due to Impaired Transportin Function. Cell Rep 24:987-1000.e7 (2018). Read more (PubMed: 30044993) »
See all 30 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Application
Western blot
Loading amount
15 µg
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (4-12% Bis-Tris Gel, MES running buffer)
Sample
Human Cell lysate - whole cell (Human Embryonic Kidney)
Specification
Human Embryonic Kidney
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Mar 18 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
Sample
Rat Cell (oligodendrocytes)
Specification
oligodendrocytes
Permeabilization
Yes - 0,1%Triton X100
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jan 03 2014

Answer

I have not been able to find the Drosophila sequence to check homology of the immunogen for either of these antibodies (full-length human hnRNP-A1) with the Drosophila sequence. This allows us to assess the likelihood of cross-reaction of the antibody with the untested species. I did find an apparent analog, the product of the Dm gene Hrb87F (Synonym: hrp36). Alignment of human hnRNP1 with this protein returns 57% identity. This suggests low potential for cross-reactivity (we look for 85% or better), but if your customer is still interested in trying one of these to IP Dm protein, I suggest ab5832, as there have been inconsistent results lately from ab10685. However, the antibody does not IP the hnRNP complex.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (NSC34)
Loading amount
10 µg
Specification
NSC34
Gel Running Conditions
Reduced Denaturing (4-12% nupage gel)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 13 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (GM10725 Lymphoblastoid cell line)
Specification
GM10725 Lymphoblastoid cell line
Fixative
Paraformaldehyde
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 5%

Abcam user community

Verified customer

Submitted Apr 02 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (cell type: IDH4)
Loading amount
5 µg
Specification
cell type: IDH4
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10%

Miss. Elizabeth Kovacs

Verified customer

Submitted Jun 06 2006

Question

BATCH NUMBER 132644 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM no band detectable even after an exposure time of 20 min SAMPLE protein extract derived from peripheral blood mononuclear cells (PBMCs) PRIMARY ANTIBODY protein transfer to nitrocellulose membrane by wet blotting (over-night, 30V), standard transfer buffer based on methanol, after transfer: quality control by Ponceau-staining, blocking of membrane with 6% milk/TBS-Tween (based on non-fat dry milk) for 4 hours @ RT DETECTION METHOD SuperSignal West Pico Chemiluminescent Substrate (Pierce), detection with Hyperfilm ECL (Amersham) POSITIVE AND NEGATIVE CONTROLS USED the same membrane was stained with antibodies against beta-actin and SF2/ASF which worked great as usual; furthermore, the same protein extracts were used to stain SMN; hnRNP A1 is ubiquitously expressed and should therefore also be present in PBMCs; a positive control was not delivered with the antibody ANTIBODY STORAGE CONDITIONS as indicated SAMPLE PREPARATION isolation of PBMCs using BD Vacutainer? CPT Cell Preparation tubes, 2x washing of cells with 1xPBS, protein extraction with RIPA w/o protease inhibitors, sample storage @-80?C; volume corresponding to 7.5?g of protein/sample plus 2xLaemmli buffer, heating @95?C for 5 min, 12%SDS PAGE @50-100V for about 2.5h TRANSFER AND BLOCKING CONDITIONS protein transfer to nitrocellulose membrane by wet blotting (over-night, 30V), standard transfer buffer based on methanol, after transfer: quality control by Ponceau-staining, blocking of membrane with 6% milk/TBS-Tween (based on non-fat dry milk) for 4 hours @ RT SECONDARY ANTIBODY Peroxidase-conjugated Affiniti Pure Goat Anti-Mouse IgG (H+L)(competitor) 1:10.000 in 1.5% milk/TBS Tween (based on non-fat dry milk), @4?C, for 1h Washing: 5x5 min with TBS Tween HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? An opportunity for alteration would be the use of higher amounts of the primary antibody, however, the total volume delivered is as low as 50 ?l. In our experimental set up, we used a 1:1000 dilution (10?l of antibody in 10ml total incubation volume). Switching to a 1:200 or 1:100 dilution is not possible with an antibody volume of 50?l delivered in total. Thus, alterations are only theoretically possible. ADDITIONAL NOTES the lab and me as investigator are experienced in the field of western blotting. no bands at all even after an exposure time of 20 min (besides of medium background which is present all over the blot) We would appreciate being reimbursed by another antibody aliquot of the same or another clone to hnRNP A1 which is working properly or by not paying for the aliquot we already got. A picture showing the result after 20 min exposure is available on request. Thank you very much for your efforts and your understanding!

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Answer

Thank you for your enquiry. I am sorry to hear that your customer has been having problems with ab5832. Please thank them for taking the time to fill out our questionnaire. I have read their questionnaire and I have a few comments. Your protocol is not dissimilar to one that I would recommend for this antibody. I would also recommend a RIPA extraction given hnRNP A1 is a nuclear protein. We have had a favourable review posted recently for this antiserum by Naoko Tanese. His review did not appear to indicate anything that was performed differently from the standard conditions. However, given your protocol please may I suggest that you repeat this experiment, extracting your cells once again, just to rule out the fact that hnRNP A1 was not extracted by the RIPA buffer. It would also be of benefit to prolong the incubation time to overnight at 4oC and increasing the total mass of protein loaded onto the gel to 20ug. Also if possible change from Milk as a blocker to 5% BSA as this can give cleaner results. I think that keeping the dilution at 1:1000 is Ok as again Naoko Tanese, in his review obtained good results with this dilution. I think that combining these conditions would serve to give you the best chance of obtaining some favourable results. I look forward to hearing from you.

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