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HEXIM1 (D5Y5K) Rabbit mAb (PE Conjugate) #97099
Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with JQ1 (500nM, 72 hours; green), using HEXIM1 (D5Y5K) Rabbit mAb (PE Conjugate).Learn more about how we get our images
Gallery: HEXIM1 (D5Y5K) Rabbit mAb (PE Conjugate) #97099
Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- 16% Formaldehyde (methanol free).
- 100% methanol.
- Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
- Fix for 15 min at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.
- Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
- Incubate 30 min on ice.
- Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
- Aliquot desired number of cells into tubes or wells.
- Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 1 hr at room temperature. Protect from light.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).
E. Optional DNA Dye
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted July 2009
revised June 2017
HEXIM1 (D5Y5K) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total HEXIM1 protein.Species Reactivity: Human, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly63 of human HEXIM1 protein.
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated HEXIM1 (D5Y5K) Rabbit mAb #12604.
Hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) was originally identified in vascular smooth muscle cells as a protein that is upregulated upon treatment with the differentiating agent hexamethylene bisacetamide (1). HEXIM1 binds 7SK RNA, a highly abundant non-coding RNA, and together they act as a potent inhibitor of positive transcription elongation factor b (P-TEFb) (2,3). P-TEFb phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II and is an important regulator of transcription elongation (4-8). 7SK RNA-bound HEXIM1 interacts with the cyclin T1 subunit of P-TEFb, sequestering P-TEFb in an inactive form leading to transcription inhibition (2,3). The regulation of the relative ratio of inactive to active P-TEFb in the cell by HEXIM1/7SK RNA is thought to play a critical role in regulation of a wide range of cellular gene expression programs such as estrogen and glucocorticoid receptor regulated genes (9-12).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc.