Anti-hnRNP A1 antibody (ab4791)

Western blot - hnRNP A1 antibody (ab4791)

All lanes : Anti-hnRNP A1 antibody (ab4791) at 1/500 dilution

Lane 1 : HeLa Whole Cell Extract
Lane 2 : HeLa Nuclear Extract

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/2000 dilution

Predicted band size : 41 kDa
Observed band size : 39 kDa (why is the actual band size different from the predicted?)

Immunoprecipitation - Anti-hnRNP A1 antibody (ab4791)

hnRNP A1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to hnRNP A1 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 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab4791.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 36kDa: hnRNP A1.

Immunocytochemistry/ Immunofluorescence - hnRNP A1 antibody (ab4791)

Immunofluorescent imaging of human cells (U2OS) with ab4791 reveals
the expected ribonucleoprotein particular staining in the nucleus.

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 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody for 30 minutes. All blocking and incubation steps carried out at 37 degrees. Nuclei counterstained with Hoechst stain (blue).

Luke Hughes-Davies and Rhiannon Jade, Gurdon Institute, Cambridge, UK

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - hnRNP A1 antibody (ab4791)

IHC image of hnRNP A1 staining in human skin FFPE section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab4791, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.