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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Mk Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with JQ1 (500nM, 72 hours; green), using HEXIM1 (D5Y5K) Rabbit mAb (PE Conjugate).

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Flow Cytometry

Flow cytometric analysis of HeLa cells using HEXIM1 (D5Y5K) Rabbit mAb (PE Conjugate) (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (red).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. 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

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

HEXIM1 (D5Y5K) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total HEXIM1 protein.


Species Reactivity: Human, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly63 of human HEXIM1 protein.

Product Description

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).


1.  Ouchida, R. et al. (2003) Genes Cells 8, 95-107.

2.  Michels, A.A. et al. (2004) EMBO J 23, 2608-19.

3.  Yik, J.H. et al. (2003) Mol Cell 12, 971-82.

4.  Buratowski, S. (2009) Mol Cell 36, 541-6.

5.  Lenasi, T. and Barboric, M. RNA Biol 7, 145-50.

6.  Pirngruber, J. et al. (2009) Cell Cycle 8, 3636-42.

7.  Wada, T. et al. (1998) EMBO J 17, 7395-403.

8.  Yamada, T. et al. (2006) Mol Cell 21, 227-37.

9.  Peterlin, B.M. et al. (2012) Wiley Interdiscip Rev RNA 3, 92-103.

10.  Ketchart, W. et al. (2011) Oncogene 30, 3563-9.

11.  Ogba, N. et al. (2008) Cancer Res 68, 7015-24.

12.  Shimizu, N. et al. (2005) Proc Natl Acad Sci U S A 102, 8555-60.


Entrez-Gene Id 10614
Swiss-Prot Acc. O94992


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.

97099
HEXIM1 (D5Y5K) Rabbit mAb (PE Conjugate)