Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling PMS2 with ab110638, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on human colon. The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

All lanes : Anti-PMS2 antibody [EPR3947] (ab110638) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PMS2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 96 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab110638 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab110638 was shown to react with PMS2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261776 (knockout cell lysate ab257142) was used. Wild-type HeLa and PMS2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab110638 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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 20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 0.1% TritonX-100-fixed, DAPI permeabilized Raji (human burkitt's lymphoma b lymphocyte) cells labelling PMS2 with ab110638 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in Raji cells. 4% Paraformaldehyde was used to counterstain tubulin at  dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Overlay histogram showing HeLa cells stained with ab110638 (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 (ab110638, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

ab110638 at 1/100 dilution staining PMS2 in Human colonic adenocarcinoma by Immunohistochemistry, Paraffin-embedded tissue.

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

All lanes :

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PMS2 knockout HAP1 whole cell lysate

Lysates/proteins at 30 µg per lane.

Predicted band size: 96 kDa

Lanes 1-2: Merged signal (red and green). Green - ab110638 observed at 120 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab110638 was shown to specifically react with PMS2 in wild-type HAP1 cells. No band was observed when PMS2 knockout samples were used. Wild-type and PMS2 knockout samples were subjected to SDS-PAGE.  Ab110638 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

Anti-PMS2 antibody [EPR3947] (ab110638) at 1/1500 dilution + HeLa (Human cervix adenocarcinoma epithelial cell) Whole cell lysates at 15 µg

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

Predicted band size: 96 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

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

Exposure time: 50 seconds

All lanes : Anti-PMS2 antibody [EPR3947] (ab110638) at 1/1000 dilution

Lane 1 : Jurkat cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : SH SY5Y cell lysate
Lane 4 : SKBR3 cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 96 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

Purified ab110638 at 1/50 dilution (2µg) immunoprecipitating PMS2 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab110638 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab110638 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/5000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 110 kDa
Lower bands are degradation bands and fresh lysate is recommended.

Purified ab110638 at 1/50 dilution (2µg) immunoprecipitating PMS2 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab110638 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab110638 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/5000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 110 kDa
Lysate were made freshly and used in IP test immediately to minimize protein degradation. Incubation time was 2h.

 

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ab110638 at 1/100 dilution staining PMS2 in HeLa cells by Immunofluorescence.

Equilibrium disassociation constant (K_D)
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