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HSF1 (D3L8I) Rabbit mAb (PE Conjugate) #63371
Gallery: HSF1 (D3L8I) Rabbit mAb (PE Conjugate) #63371
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
HSF1 (D3L8I) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total HSF1 protein. This antibody is not predicted to cross-react with HSF2.Species Reactivity: Human, Mouse, Rat, Monkey, Bovine, Dog, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human HSF1 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 HSF1 (D3L8I) Rabbit mAb #12972.
All organisms respond to increased temperatures and other environmental stresses by rapidly inducing the expression of highly conserved heat shock proteins (HSPs) that serve as molecular chaperones to refold denatured proteins and promote the degradation of damaged proteins. Heat shock gene transcription is regulated by a family of heat shock factors (HSFs), transcriptional activators that bind to heat shock response elements (HSEs) located upstream of all heat shock genes (1). HSEs are highly conserved among organisms and contain multiple adjacent and inverse iterations of the pentanucleotide motif 5'-nGAAn-3'. HSFs are less conserved and share only 40% sequence identity. Vertebrate cells contain four HSF proteins: HSF1, 2 and 4 are ubiquitous, while HSF3 has only been characterized in avian species. HSF1 induces heat shock gene transcription in response to heat, heavy metals, and oxidative agents, while HSF2 is involved in spermatogenesis and erythroid cell development. HSF3 and HSF4 show overlapping functions with HSF1 and HSF2. The inactive form of HSF1 exists as a monomer that localizes to both the cytoplasm and nucleus, but does not bind DNA (1,2). In response to stress, HSF1 becomes phosphorylated, forms homotrimers, binds DNA and activates heat shock gene transcription (1,2). HSF1 activity is positively regulated by phosphorylation of Ser419 by PLK1, which enhances nuclear translocation, and phosphorylation of Ser230 by CaMKII, which enhances transactivation (3,4). Alternatively, HSF1 activity is repressed by phosphorylation of serines at 303 and 307 by GSK3 and ERK1, respectively, which leads to binding of 14-3-3 protein and sequestration of HSF1 in the cytoplasm (5,6). In addition, during attenuation from the heat shock response, HSF1 is repressed by direct binding of Hsp70, HSP40/Hdj-1, and HSF binding protein 1 (HSBP1) (7).
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.