Anti-CHMP2B 抗体

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CHMP2B antibody (AB33174)

ab33174 staining CHMP2B in A431 cells. The cells were fixed with 100% methanol (5 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 ab33174 at 5µg/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).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Project1613****

Western blot - Anti-CHMP2B antibody (AB33174)

ab33174 was tested on the full length recombinant tagged CHMP2B protein which is predicted to run at 50 kDa.

All lanes:

Western blot - Anti-CHMP2B antibody (ab33174) at 1 µg/mL

All lanes:

Tagged recombinant CHMP2B protein at 0.1 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 24 kDa

Observed band size: 55 kDa

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• WB

Lab

Western blot - Anti-CHMP2B antibody (AB33174)

Lanes 1 - 4 : Merged signal (red and green). Green - ab33174 observed at 30 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab33174 was shown to recognize CHMP2B in wild-type A549 cells as signal was lost at the expected MW in CHMP2B knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CHMP2B knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab33174 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml 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-CHMP2B antibody (ab33174) at 1 µg/mL

Lane 1:

Wild-type A549 (Human lung carcinoma cell line) 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='/products/cell-lines/human-chmp2b-knockout-a549-cell-line-ab261874'>ab261874</a>)

Lane 3:

A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

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

Predicted band size: 24 kDa

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• WB

CiteAb

Western blot - Anti-CHMP2B antibody (AB33174)

CHMP2B western blot using anti-CHMP2B antibody ab33174. Publication image and figure legend from Kumar, H., Pushpa, K., et al., 2019, J Cell Sci, PubMed 31221728.

ab33174 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab33174 please see the product overview.

The exocyst complex and Rab5 deliver ESCRT III subunits to the cytokinetic bridge. (A–C) Representative confocal micrographs of HeLa cells treated with control (GFP), Exoc3, Exoc4 and Rab5 siRNAs, fixed and stained for α-tubulin (red), CHMP2B (green, A,C), CHMP4B (green, B) and chromatin (DAPI, blue). Scale bars : 10 μm. The area boxed in white is magnified in the image on the right, depicting the midbody ring and secondary constriction region of the cytokinetic bridge. The representative white lines placed beside the cytokinetic bridges (zoom) indicate the corresponding regions within the bridge that were used for line-scan intensity analysis. The lines were of a constant length of 2.32 μm, with the end points coinciding with the secondary constrictions visualized using α-tubulin (microtubule) staining. (D) Representative line-scan profile for CHMP2B intensity (y-axis) upon control (siGFP) and Exoc3 (siExoc3) depletion. (E,F) Quantification of mean fluorescence intensity of CHMP2B (E) and CHMP4B (F) at the mid-body region using linescan tool from 20 cells across three independent experiments, shown as graphs with mean±s.d. *p<0.05, **p<0.01, ***p<0.001. (G) Immunoblots showing depletion of the indicated proteins (IB); α-tubulin is shown as a loading control. (H) Immunoblots probing for pulldown of CHMP2B upon immunoprecipitation of EGFP–Rab5 or EGFP. The blot was probed with the respective antibodies as indicated (IB).

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