All lanes : Anti-DPF2/REQ antibody [EPR9206(B)] (ab134942) at 1/1000 dilution

Lane 1 : Wild-type A431 cell lysate
Lane 2 : DPF2 knockout A431 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : LNCaP cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 44 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab134942 observed at 50 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab134942 was shown to react with DPF2/REQ in wild-type A431 cells in western blot. Loss of signal was observed when DPF2 knockout sample was used. Wild-type and DPF2 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab134942 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab134942 (purified) at 1/100 dilution (2µg) immunoprecipitating DPF2/REQ in HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate.

Lane 1 (input): HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate 10ug
Lane 2 (+): ab134942 + HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab134942 in HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling DPF2/REQ with purified ab134942 at 1/200 dilution (8.3μg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889, an anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/ml). ab150077, a Goat anti-rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1/1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue sections labeling DPF2/REQ with purified ab134942 at 1/200 dilution (8.3 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH9. Tissue was counterstained with hematoxylin. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebral cortex tissue sections labeling DPF2/REQ with purified ab134942 at 1/200 dilution (8.3 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH9. Tissue was counterstained with hematoxylin. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebral cortex tissue sections labeling DPF2/REQ with purified ab134942 at 1/200 dilution (8.3 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH9. Tissue was counterstained with hematoxylin. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling DPF2/REQ with purified ab134942 at 1/200 dilution (8.3 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH9. Tissue was counterstained with hematoxylin. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

Anti-DPF2/REQ antibody [EPR9206(B)] (ab134942) at 1/2000 dilution (purified) + NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 44 kDa
Observed band size: 44 kDa

Blocking and diluting buffer: 5% NFDM /TBST.

All lanes : Anti-DPF2/REQ antibody [EPR9206(B)] (ab134942) at 1/1000 dilution (purified)

Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : LNCaP (Human prostate cancer cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 44 kDa
Observed band size: 44 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

All lanes : Anti-DPF2/REQ antibody [EPR9206(B)] (ab134942) at 1/1000 dilution (unpurified)

Lane 1 : HeLa cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : LNCaP cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Standard HRP labelled goat anti-rabbit at 1/2000 dilution

Developed using the ECL technique.

Predicted band size: 44 kDa

Immunohistochemical analysis of paraffin-embedded, formalin-fixed Human kidney tissue, labelling DPF2/REQ using unpurified ab134942 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Equilibrium disassociation constant (K_D)
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