Anti-Hamartin antibody [EPR24364-109] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Hamartin antibody [EPR24364-109] - BSA and Azide free (AB282016)

ab270967 staining Hamartin in wild-type HAP1 cells (top panel) and TSC1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab270967 at 0.2µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). The antibody is not suitable to detect Hamartin in cells fixed with 100% methanol (5 min). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Hamartin antibody [EPR24364-109] - BSA and Azide free (AB282016)

This data was developed using ab270967, the same antibody clone in a different buffer formulation.

ab270967 was shown to react with TSC1 in wild-type HAP1 cells in immunocytochemistry with loss of signal observed in a TSC1 knockout cell line. ab270967 at  1/500 dilution.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• IP

Supplier Data

Immunoprecipitation - Anti-Hamartin antibody [EPR24364-109] - BSA and Azide free (AB282016)

This data was developed using ab270967, the same antibody clone in a different buffer formulation.

Hamartin was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab270967 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270967 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug

Lane 2 : ab270967 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab270967 in HeLa whole cell lysate

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-Hamartin antibody [EPR24364-109] (<a href='/en-us/products/primary-antibodies/hamartin-antibody-epr24364-109-ab270967'>ab270967</a>)

Predicted band size: 130 kDa

Observed band size: 150 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-Hamartin antibody [EPR24364-109] - BSA and Azide free (AB282016)

This data was developed using ab270967, the same antibody clone in a different buffer formulation.

Hamartin was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab270967 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270967 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 ug

Lane 2 : ab270967 IP in NIH/3T3 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab270967 in NIH/3T3 whole cell lysate

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-Hamartin antibody [EPR24364-109] (<a href='/en-us/products/primary-antibodies/hamartin-antibody-epr24364-109-ab270967'>ab270967</a>)

Predicted band size: 130 kDa

Observed band size: 150 kDa

false

• WB

Lab

Western blot - Anti-Hamartin antibody [EPR24364-109] - BSA and Azide free (AB282016)

Western blot : Anti-TSC1 antibody [EPR24364-109] (ab270967) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab270967 was shown to bind specifically to TSC1. A band was observed at 140-150 kDa in wild-type MCF7 cell lysates with no signal observed at this size in TSC1 knockout cell line. To generate this image, wild-type and TSC1 knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Hamartin antibody [EPR24364-109] (<a href='/en-us/products/primary-antibodies/hamartin-antibody-epr24364-109-ab270967'>ab270967</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

TSC1 knockout MCF7 cell lysate at 20 µg

Lane 3:

Wild-type A549 cell lysate at 20 µg

Lane 4:

TSC1 knockout A549 cell lysate at 20 µg

Lane 5:

Wild-type HAP1 cell lysate at 20 µg

Lane 6:

TSC1 knockout HAP1 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

false

• WB

Lab

Western blot - Anti-Hamartin antibody [EPR24364-109] - BSA and Azide free (AB282016)

This data was developed using ab270967, the same antibody clone in a different buffer formulation.

ab270967 was shown to react with TSC1 in wild-type HAP1 cells in Western blot with loss of signal observed in a TSC1 knockout cell line. Wild-type HAP1 and TSC1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab270967 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Hamartin antibody [EPR24364-109] (<a href='/en-us/products/primary-antibodies/hamartin-antibody-epr24364-109-ab270967'>ab270967</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 100 µg

Lane 2:

TSC1 knock-out HAP1 lysate at 100 µg

false

• WB

Lab

Western blot - Anti-Hamartin antibody [EPR24364-109] - BSA and Azide free (AB282016)

This data was developed using ab270967, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lanes 1-3 : Merged signal (red and green).

Green - ab270967 observed at 150 kDa.

Red - loading control ab8245 observed at 36 kDa.

ab270967 Anti-Hamartin antibody [EPR24364-109] was shown to specifically react with Hamartin in wild-type HAP1 cells. Loss of signal was observed when the knockout cell line was used. Wild-type and Hamartin knockout samples were subjected to SDS-PAGE. ab270967 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1/1000 dilution and 1/20000 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Hamartin antibody [EPR24364-109] (<a href='/en-us/products/primary-antibodies/hamartin-antibody-epr24364-109-ab270967'>ab270967</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

Hamartin knockout HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 130 kDa

Observed band size: 150 kDa

false

• WB

Lab

Western blot - Anti-Hamartin antibody [EPR24364-109] - BSA and Azide free (AB282016)

This data was developed using ab270967, the same antibody clone in a different buffer formulation.

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

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

Exposure time : 48 seconds.

All lanes:

Western blot - Anti-Hamartin antibody [EPR24364-109] (<a href='/en-us/products/primary-antibodies/hamartin-antibody-epr24364-109-ab270967'>ab270967</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 130 kDa

Observed band size: 150 kDa

false