Anti-CMT2 antibody [EPR9584]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CMT2 antibody [EPR9584] (AB150363)

Immunohistochemical analysis of paraffin embedded Human brain tissue labelling CMT2 binding protein with ab150363 antibody at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CMT2 antibody [EPR9584] (AB150363)

ab150363 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab150363 at 5μg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CMT2 antibody [EPR9584] (AB150363)

Immunohistochemical analysis of paraffin embedded Human cervical carcinoma tissue labelling CMT2 binding protein with ab150363 antibody at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• WB

Unknown

Western blot - Anti-CMT2 antibody [EPR9584] (AB150363)

All lanes:

Western blot - Anti-CMT2 antibody [EPR9584] (ab150363) at 1/1000 dilution

Lane 1:

HepG2 cell lysate at 10 µg

Lane 2:

A431 cell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 31 kDa

Observed band size: 34 kDa

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• WB

Lab

Western blot - Anti-CMT2 antibody [EPR9584] (AB150363)

False colour image of Western blot : Anti-CMT2 antibody [EPR9584] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab150363 was shown to bind specifically to CMT2. A band was observed at 35 kDa in wild-type HeLa cell lysates with no signal observed at this size in MAD2L1BP knockout cell line ab265854 (knockout cell lysate ab257510). To generate this image, wild-type and MAD2L1BP knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-CMT2 antibody [EPR9584] (ab150363) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Human MAD2L1BP (CMT2) knockout HeLa cell lysate (<a href='/en-us/products/unavailable/human-mad2l1bp-cmt2-knockout-hela-cell-lysate-ab257510'>ab257510</a>) at 20 µg

Lane 2:

Western blot - Human MAD2L1BP (CMT2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-mad2l1bp-cmt2-knockout-hela-cell-line-ab265854'>ab265854</a>)

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Lane 5:

MDA-MB-231 cell lysate at 20 µg

Predicted band size: 31 kDa

Observed band size: 35 kDa

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• WB

CiteAb

Western blot - Anti-CMT2 antibody [EPR9584] (AB150363)

CMT2 western blot using anti-CMT2 antibody [EPR9584] ab150363. Publication image and figure legend from de Lange, J., Faramarz, A., et al., 2015, Nat Commun, PubMed 26423134.

ab150363 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 ab150363 please see the product overview.

Defective sister chromatid cohesion sensitizes to APC/C inhibition.(a) Cohesion defect analysis of five tumour cell lines (b,c) Cells were transfected with the indicated siRNAs. Protein levels were analysed after three days by western blot and cell viability was measured after 5 days using a CellTiter-Blue assay. Error bars denote s.d. of at least three technical replicates. (d,e) A panel of tumour cell lines with known cohesion status was seeded at optimized densities in two 96-wells plates. The next day, viability was assayed in one plate (t=0), whereas in the other plate medium was replaced with medium containing DMSO, apcin (150 μM) or paclitaxel (2 nM) and viability was measured 3 days later. We calculated the percentage growth inhibition of treated versus untreated cells and corrected this for the number of cell divisions (indicated between brackets) in untreated cells during the experiment. Error bars denote s.d. of at least three technical replicates. Apcin sensitivity was significantly higher (P=0.030) in cohesion-defective cells as compared with cells without cohesion defects, whereas paclitaxel sensitivity did not differ (P=0.537) using a Wilcoxon rank-sum test.

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