Anti-HEXIM1 antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HEXIM1 antibody (AB25388)

ab25388 staining HEXIM1 in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 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 ab25388 at 1µg/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.

• IP

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Immunoprecipitation - Anti-HEXIM1 antibody (AB25388)

HEXIM1 - ChIP Grade was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to HEXIM1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab25388.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 54kD : HEXIM1 .

All lanes:

Immunoprecipitation - Anti-HEXIM1 antibody (ab25388)

Predicted band size: 41 kDa

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• WB

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Western blot - Anti-HEXIM1 antibody (AB25388)

ab25388 recognizes a band at approximately 54 kDa that corresponds in size to that seen for HEXIM1. Although it has a predicted molecular weight of 41 kDa, it has been shown to migrate at a larger size of about 54-60 kDa (see Byers et al., J Biol Chem. 2005 Apr 22;280(16) : 16360-7 and Schulte et al., J. Biol. Chem., Vol. 280, (26) : 24968-24977).

All lanes:

Western blot - Anti-HEXIM1 antibody (ab25388) at 1/250 dilution

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 3:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 5:

SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 41 kDa

Observed band size: 54 kDa

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