Anti-CHMP2B antibody [EPR10807(B)]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CHMP2B antibody [EPR10807(B)] (AB157208)

Immunocytochemistry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling CHMP2B with Purified ab157208 at 1 : 250 dilution (0.6 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CHMP2B antibody [EPR10807(B)] (AB157208)

ab157208 was shown to react with CHMP2B in wild-type U2OS cells in immunocytochemistry with loss of signal observed in a CHMP2B knockout cell line. 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 ab157208 at 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 ?g/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).

This data was 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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CHMP2B antibody [EPR10807(B)] (AB157208)

Flow Cytometry analysis of 293T (Human embryonic kidney epithelial cell) cells labelling CHMP2B with Purified ab157208 at 1 : 20 dilution (10 µg/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). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• WB

Lab

Western blot - Anti-CHMP2B antibody [EPR10807(B)] (AB157208)

ab157208 was shown to react with CHMP2B in wild-type U2OS cells in Western blot with loss of signal observed in a CHMP2B knockout cell line. Wild-type U2OS and CHMP2B knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab157208 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2ug/mL before imaging.

This data was 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.

Lanes 1 - 2 : Western blot – Anti-CHMP2B antibody [EPR10807(B)] (ab157208) at 1/1000 dilution

Lane 1 : Wild-type U2OS lysate at 20 g

Lane 2 : CHMP2B knockout U2OS cell lysate at 20 µg

Performed under reducing conditions

All lanes:

Western blot - Anti-CHMP2B antibody [EPR10807(B)] (ab157208) at 1/1000 dilution

Lane 1:

Wild-type U2OS lysate at 20 µg

Lane 2:

CHMP2B knock-out U2OS lysate at 24 µg

Observed band size: 24 kDa

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• IP

Supplier Data

Immunoprecipitation - Anti-CHMP2B antibody [EPR10807(B)] (AB157208)

Purified ab157208 at 1 : 20 dilution (0.7μg) immunoprecipitating CHMP2B in 293T whole cell lysate.

Lane 1 (input) : 293T (Human embryonic kidney epithelial cell) whole cell lysate 10μg.
Lane 2 (+) : ab157208 + 293T whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab157208 in 293T whole cell lysate.
VeriBlot for IP Detection Reagent (HRP)(ab131366) (1 : 1000 dilution) was used for Western blotting.

Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 30 kDa
We are unsure about the nature of the 27kDa band. It may be isoform 2 of CHMP2B.

All lanes:

Immunoprecipitation - Anti-CHMP2B antibody [EPR10807(B)] (ab157208)

Predicted band size: 24 kDa

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• WB

Unknown

Western blot - Anti-CHMP2B antibody [EPR10807(B)] (AB157208)

We are unsure about the nature of the 27kDa band. It may be isoform 2 of CHMP2B.

All lanes:

Western blot - Anti-CHMP2B antibody [EPR10807(B)] (ab157208) at 1/1000 dilution

Lane 1:

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

Lane 2:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

A549 (Human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

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

Lane 5:

Mouse brain lysate at 20 µg

Lane 6:

Mouse heart lysate at 20 µg

Lane 7:

Rat brain lysate at 20 µg

Lane 8:

Rat heart 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: 24 kDa

Observed band size: 30 kDa

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• WB

Lab

Western blot - Anti-CHMP2B antibody [EPR10807(B)] (AB157208)

Lanes 1 - 4 : Merged signal (red and green). Green - ab157208 observed at 45 kDa. Red - loading control, ab8245, observed at 38 kDa.

ab157208 was shown to specifically react with CHMP2B in wild-type A549 cells as signal was lost in CHMP2B knockout cells. Wild-type and CHMP2B knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab157208 and ab8245 (Mouse monoclonal [6C5] to GAPDH - Loading Control) were incubated overnight at 4°C at 1/1000 dilution and 1/1000 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-CHMP2B antibody [EPR10807(B)] (ab157208) at 1/1000 dilution

Lane 1:

Wild-type A549 whole cell lysate at 20 µg

Lane 2:

CHMP2B knockout A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CHMP2B knockout A549 cell line (<a href='/en-us/products/cell-lines/human-chmp2b-knockout-a549-cell-line-ab261874'>ab261874</a>)

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 24 kDa

false