Anti-GAPDH Antibody - (ab8245) | Danaher Life Sciences

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

Lanes 1- 2: Merged signal (red and green). Green - ab16640 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab16640 was shown to react with Sortilin/NT3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264772 (knockout cell lysate ab257696) was used. Wild-type HeLa and SORT1 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. ab16640 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 μg/ml 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.

All lanes:

Western blot - Anti-Sortilin/NT3 antibody (ab16640) at 1 µg/mL

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate (ab257696) at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at 1/20000 dilution

Performed under reducing conditions.

Predicted band size: 92 kDa

Observed band size: 100 kDa

Western blot analysis of Sortilin/NT3 (ab16640) expression in wild-type and SORT1 knockout HeLa cells.

Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

ab8245 staining GAPDH in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed with 100% methanol (5 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab8245 at 5 μg/ml and ab6046 at 1 μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) at 2 μg/ml (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (ab150088) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.

Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

HeLa cells stained with ab8245 (GAPDH) and DAPI (nuclear DNA), showing specific staining and negative controls for unspecific antibody reactions.

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab140751 overnight at 4°C. Antibody binding was detected using the Donkey Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216779 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

All lanes:

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/2000 dilution

Lane 1:

Wild-type HAP1 cell lysate (20 µg)

Lane 2:

NF-κB p60 knockout HAP1 cell lysate (20 µg)

Lane 3:

HeLa cell lysate (20 µg)

Lane 4:

A431 cell lysate (20 µg)

Secondary

All lanes:

Western blot - Donkey Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216779) at 1/10000 dilution

Predicted band size: 36 kDa

Western blot analysis of GAPDH expression in various cell lines.

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

False colour image of Western blot: Anti-SIRPA antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to SIRPA. A band was observed at 100-140 kDa (mouse SIRPA, isoform 1), in wild-type RAW 264.7 cell lysates (band observed at 70-100 kDa in THP-1 is Human SIRPA) with no signal observed at this size in SIRPA knockout cell line ab281618 (knockout cell lysate ab282969). To generate this image, wild-type and SIRPA knockout RAW 264.7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/20000 dilution

Lane 1:

Wild-type RAW 264.7 cell lysate at 20 µg

Lane 2:

RAW 264.7 cell lysate at 20 µg

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Predicted band size: 55 kDa

Western blot analysis of Sortilin/NT3 protein expression in wild-type and SORT1 knockout HeLa cells using ab16640 antibody.

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

Lane 1: Wild-type HeLa cell lysate (20µg)

Lane 2: SORT1 knockout HeLa cell lysate (20µg)

Lanes 1- 2: Merged signal (red and green). Green - ab188586 observed at 100 kDa. Red - loading control ab8245 observed at 37 kDa.

ab188586 Anti-Sortilin/NT3 antibody [EPR15010] was shown to specifically react with Sortilin/NT3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264772 (knockout cell lysate ab257696) was used. Wild-type and Sortilin/NT3 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab188586 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (ab188586) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate (ab257696) at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at 1/20000 dilution

Performed under reducing conditions.

Predicted band size: 92 kDa

Observed band size: 100 kDa

Western blot image showing the specific reaction of anti-Sortilin/NT3 antibody (ab188586) with Sortilin/NT3 protein in wild-type HeLa cells, with a loss of signal in SORT1 knockout HeLa cells.