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

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.

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.

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.

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).

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 Detection Reagent (ab131366) was used for detection (1/10 000). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

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?

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.

All lanes : Anti-Hsc70 antibody [EP1531Y] (ab51052) at 1/5000 dilution (purified)

Lane 1 : C6 cell lysate
Lane 2 : PC-12 cell lysate
Lane 3 : Mouse heart tissue lysate
Lane 4 : Rat heart tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

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

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Hsc70 antibody [EP1531Y] (ab51052) at 1/5000 dilution (purified)

Lane 1 : human fetal brain lysate
Lane 2 : HeLa cell lysate
Lane 3 : HEK293 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

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

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST