Western blot - HEXIM1 antibody - ChIP Grade (ab25388)

All lanes : Anti-HEXIM1 antibody - ChIP Grade (ab25388) at 1/250 dilution

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 5 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size : 41 kDa
Observed band size : 54 kDa (why is the actual band size different from the predicted?)


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).

Immunoprecipitation - Anti-HEXIM1 antibody - ChIP Grade (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 .

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HEXIM1 antibody - ChIP Grade (ab25388)

Image courtesy of Human Protein Atlas

ab25388 staining HEXIM1 in human kidney. Paraffin embedded human kidney tissue was incubated with ab25388 (1/100 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

ab25388 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines.

Further results for this antibody can be found at www.proteinatlas.org

Immunocytochemistry/ Immunofluorescence - Anti-HEXIM1 antibody - ChIP Grade (ab25388)

ICC/IF image of ab25388 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 ab25388 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 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.