重组Anti-Transglutaminase 2抗体[EP2957]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Transglutaminase 2 antibody [EP2957] (AB109200)

ab109200 was shown to react with TGM2 in wild-type A549 cells in immunocytochemistry with loss of signal observed in TGM2 knockout cell line ab261876. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab109200 at 1/300 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 µg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Transglutaminase 2 antibody [EP2957] (AB109200)

ab109200, at 1/100 dilution, staining Transglutaminase 2 in paraffin-embedded Human kidney tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-Transglutaminase 2 antibody [EP2957] (AB109200)

ab109200 was shown to react with TGM2 in wild-type A549 cells in Western blot with loss of signal observed in TGM2 knockout cell line ab261876. Wild-type A549 and TGM2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab109200 overnight at 4 °C at a 1/10000 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-Transglutaminase 2 antibody [EP2957] (ab109200) at 1/10000 dilution

Lane 1:

Wild-type A549 lysate at 40 µg

Lane 2:

Western blot - Human TGM2 (Transglutaminase 2) knockout A549 cell line (<a href='/products/cell-lines/human-tgm2-transglutaminase-2-knockout-a549-cell-line-ab261876'>ab261876</a>) at 40 µg

Observed band size: 75 kDa

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• WB

Lab

Western blot - Anti-Transglutaminase 2 antibody [EP2957] (AB109200)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109200 observed at 77 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109200 was shown to recognize TGM2 in wild-type A549 cells as signal was lost at the expected MW in TGM2 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and TGM2 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab109200 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Transglutaminase 2 antibody [EP2957] (ab109200) at 1/1000 dilution

Lane 1:

Wild-type A549 whole cell lysate at 20 µg

Lane 2:

TGM2 knockout A549 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

HUVEC whole cell lysate at 20 µg

Predicted band size: 77 kDa

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• WB

Unknown

Western blot - Anti-Transglutaminase 2 antibody [EP2957] (AB109200)

All lanes:

Western blot - Anti-Transglutaminase 2 antibody [EP2957] (ab109200) at 1/10000 dilution

Lane 1:

U87-MG cell lysate at 10 µg

Lane 2:

A549 cell lysate at 10 µg

Lane 3:

HUVEC cell lysate at 10 µg

Predicted band size: 77 kDa

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• WB

Lab

Western blot - Anti-Transglutaminase 2 antibody [EP2957] (AB109200)

Lanes 1-4 : Merged signal (red and green). Green - ab109200 observed at 77 kDa. Red - loading control ab8245 observed at 37 kDa.

ab109200 Anti-Transglutaminase 2 antibody [EP2957] was shown to specifically react with Transglutaminase 2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265245 (knockout cell lysate ab257085) was used. Wild-type and Transglutaminase 2 knockout samples were subjected to SDS-PAGE. ab109200 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) were incubated overnight at 4°C at 1 in 1000 dilution 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-Transglutaminase 2 antibody [EP2957] (ab109200) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

TGM2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human TGM2 (Transglutaminase 2) knockout HeLa cell line (<a href='/products/cell-lines/human-tgm2-transglutaminase-2-knockout-hela-cell-line-ab265245'>ab265245</a>)

Lane 3:

Wild-type A549 cell lysate at 20 µg

Lane 4:

TGM2 knockout A549 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 77 kDa

Observed band size: 77 kDa

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• WB

Lab

Western blot - Anti-Transglutaminase 2 antibody [EP2957] (AB109200)

Lanes 1-2 : Merged signal (red and green). Green - ab109200 observed at 77 kDa. Red - loading control ab8245 observed at 37 kDa.

ab109200 Anti-Transglutaminase 2 antibody [EP2957] was shown to specifically react with Transglutaminase 2 in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267110 (knockout cell lysate ab257087) was used. Wild-type and Transglutaminase 2 knockout samples were subjected to SDS-PAGE. ab109200 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 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-Transglutaminase 2 antibody [EP2957] (ab109200) at 1/10000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

TGM2 knockout A549 cell lysate at 20 µg

Predicted band size: 77 kDa

Observed band size: 77 kDa

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