重组Anti-Hamartin抗体[EP318Y] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-Hamartin antibody [EP318Y] - BSA and Azide free (AB247297)

This data was developed using ab40872, the same antibody clone in a different buffer formulation.

Immunoprecipitation of TSC1 in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using  2.0 µg of ab40872 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

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:

Immunoprecipitation - Anti-Hamartin antibody [EP318Y] (<a href='/products/primary-antibodies/hamartin-antibody-ep318y-ab40872'>ab40872</a>) at 2 µg

All lanes:

HAP1 cells

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• WB

Lab

Western blot - Anti-Hamartin antibody [EP318Y] - BSA and Azide free (AB247297)

This data was developed using ab40872, the same antibody clone in a different buffer formulation.

ab40872 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 ab40872 overnight at 4 °C at a 1/500 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 [EP318Y] (<a href='/products/primary-antibodies/hamartin-antibody-ep318y-ab40872'>ab40872</a>) at 1/500 dilution

Lane 1:

Wild-type HAP1 lysate at 100 µg

Lane 2:

TSC1 knock-out HAP1 lysate at 100 µg

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• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Hamartin antibody [EP318Y] - BSA and Azide free (AB247297)

This data was developed using ab40872, the same antibody clone in a different buffer formulation.Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Hamartin with Purified ab40872 at 1/20 dilution (10μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hamartin antibody [EP318Y] - BSA and Azide free (AB247297)

This data was developed using ab40872, the same antibody clone in a different buffer formulation.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human liver tissue sections labeling Hamartin with purified ab40872 at 1/200 dilution (1.08 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Unknown

Western blot - Anti-Hamartin antibody [EP318Y] - BSA and Azide free (AB247297)

We are not sure about the nature of the extra band.

This data was developed using ab40872, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-Hamartin antibody [EP318Y] (<a href='/products/primary-antibodies/hamartin-antibody-ep318y-ab40872'>ab40872</a>) at 1/1000 dilution

All lanes:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Secondary

All lanes:

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

Predicted band size: 130 kDa

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• WB

Lab

Western blot - Anti-Hamartin antibody [EP318Y] - BSA and Azide free (AB247297)

This data was developed using ab40872, the same antibody clone in a different buffer formulation.

Lane 1 : Wild-type HAP1 cell lysate (20 μg)

Lane 2 : Hamartin knockout HAP1 cell lysate (20 μg)

Lane 3 : HeLa cell lysate (20 μg)

Lane 4 : Human skeletal muscle tissue lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab40872 observed at 150 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab40872 was shown to recognize Hamartin when Hamartin knockout samples were used, along with additional cross-reactive bands. Wild-type and Hamartin knockout samples were subjected to SDS-PAGE. ab40872 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/10000 respectively and incubated overnight at 4°C. 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 [EP318Y] (<a href='/products/primary-antibodies/hamartin-antibody-ep318y-ab40872'>ab40872</a>)

Predicted band size: 130 kDa

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• WB

Lab

Western blot - Anti-Hamartin antibody [EP318Y] - BSA and Azide free (AB247297)

Western blot : Anti-TSC1 antibody [EP318Y] (ab40872) 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, ab40872 was shown to bind specifically to TSC1. A band was observed at 130-150 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 [EP318Y] (<a href='/products/primary-antibodies/hamartin-antibody-ep318y-ab40872'>ab40872</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

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• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Hamartin antibody [EP318Y] - BSA and Azide free (AB247297)

This data was developed using ab40872, the same antibody clone in a different buffer formulation.Overlay histogram showing HeLa cells stained with ab40872 (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 (ab40872, 1/10000 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. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.