All lanes : Anti-Hsc70 antibody [EP1531Y] (ab51052) at 1/500 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : HSPA8 knockout HeLa cell lysate
Lane 3 : A431 cell lysate
Lane 4 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 71 kDa
Observed band size: 71 kDa

This data was developed using the same antibody clone in a different buffer formulation (ab51052).

Lanes 1- 4: Merged signal (red and green). Green - ab51052 observed at 71 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab51052 was shown to react with Hsc70 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265664 (knockout cell lysate ab256944) was used. Wild-type HeLa and HSPA8 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. ab51052 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 500 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.

ab51052 (purified) at 1/20 immunoprecipitating Hsc70 in 10 µg HeLa (Lanes 1 and 2, observed at 71 kDa). Lane 3 - PBS. For western blotting, HRP Veriblot for IP (ab131366) was used for detection at 1/1000 dilution. Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51052).

Overlay histogram showing MCF7 cells fixed in 4% PFA and stained with purified ab51052 at a dilution of 1 in 70 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51052).

Immunofluorescence staining of MCF7 cells with purified ab51052 at a working dilution of 1/250, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab51052 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51052).

All lanes : Anti-Hsc70 antibody [EP1531Y] (ab51052) at 1/500 dilution

Lane 1 : Wild-type A431 whole cell lysate
Lane 2 : HSPA8 knockout A431 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : MCF7 whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 71 kDa
Observed band size: 73 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab51052).

Lanes 1 - 4: Merged signal (red and green). Green - ab51052 observed at 73 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab51052 was shown to react with HSPA8 in A431 wild-type cells in Western blot. Loss of signal was observed when HSPA8 knockout sample was used. A431 wild-type and HSPA8 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with ab51052 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 500 dilution and a 1 in 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical staining of paraffin embedded human cerebral cortex with purified ab51052 at a working dilution of 1/500. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51052).

Unpurified ab51052 (1/250) staining Hsc70 in paraffin embedded human breast carcinoma tissue by Immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51052).

Immunofluorescent staining of HeLa cells using unpurified ab51052 (1/100).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51052).