Anti-hnRNP A2B1 antibody (ab31645)

All lanes : Anti-hnRNP A2B1 antibody (ab31645) at 1/500 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : HNRNPA2B1 knockout HEK293T cell lysate
Lane 3 : A549 cell lysate at 20 µg

Performed under reducing conditions.

Predicted band size: 35 , 37 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab31645 observed at 37 kDa. Red - loading control, ab7291 observed at 50 kDa.

ab31645 Anti-hnRNP A2B1 antibody was shown to specifically react with hnRNP A2B1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266404 (knockout cell lysate ab257224) was used. Wild-type and hnRNP A2B1 knockout samples were subjected to SDS-PAGE. ab31645 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti- Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti- Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

 

 

ab31645 staining hnRNP A2B1 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab31645 at 5 µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Anti-hnRNP A2B1 antibody (ab31645) at 1 µg/ml + HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Secondary
Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution


Performed under reducing conditions.

Predicted band size: 35 , 37 kDa
Observed band size: 35,38 kDa why is the actual band size different from the predicted?



ab31645 is expected to recognize isoform hnrnp A2 and hnrnp B1. The antibody detects bands at approximately 35 and 38 kDa which are of the correct size to correspond to the predicted molecular weight of these isoforms.

hnRNP A2B1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to hnRNP A2B1 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 ab31645.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 35kDa: hnRNP A2B1.

IHC image of ab31645 staining in human colon formalin fixed paraffin embedded tissue 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 ab31645, 1µg/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.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.