Anti-CMT2 antibody [EPR9584] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CMT2 antibody [EPR9584] - BSA and Azide free (AB248941)

This data was developed using ab150363, the same antibody clone in a different buffer formulation.

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] - BSA and Azide free (AB248941)

This data was developed using ab150363, the same antibody clone in a different buffer formulation.

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] - BSA and Azide free (AB248941)

This data was developed using ab150363, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-CMT2 antibody [EPR9584] (<a href='/en-us/products/primary-antibodies/cmt2-antibody-epr9584-ab150363'>ab150363</a>) 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

false

• WB

Lab

Western blot - Anti-CMT2 antibody [EPR9584] - BSA and Azide free (AB248941)

This data was developed using ab150363, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/cmt2-antibody-epr9584-ab150363'>ab150363</a>) 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

false