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56831
HSP60 (D6F1) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates

HSP60 (D6F1) XP® Rabbit mAb (PE Conjugate) #56831

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Hm Mk X Z B Pg Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of HeLa cells using HSP60 (D6F1) XP® 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

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.

HSP60 (D6F1) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total HSP60 protein.

Species Reactivity:

Human, Mouse, Rat, Hamster, Monkey, Xenopus, Zebrafish, Bovine, Pig

Species predicted to react based on 100% sequence homology:

Chicken, Dog, Horse

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Trp68 of human HSP60 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 HSP60 (D6F1) XP® Rabbit mAb #12165.

In both prokaryotic and eukaryotic cells the misfolding and aggregation of proteins during biogenesis and under conditions of cellular stress are prevented by molecular chaperones (1-3). HSP60 has primarily been known as a mitochondrial protein that is important for folding key proteins after import into the mitochondria (4). Research studies have shown that a significant amount of HSP60 is also present in the cytosol of many cells, and that it is induced by stress, inflammatory and immune responses, and autoantibodies correlated with Alzheimer's, coronary artery diseases, MS, and diabetes (5-8).

  1. Hartl, F.U. (1996) Nature 381, 571-579.
  2. Bukau, B. and Horwich, A.L. (1998) Cell 92, 351-366.
  3. Hartl, F.U. and Hayer-Hartl, M. (2002) Science 295, 1852-1858.
  4. Jindal, S. et al. (1989) Mol. Cell Biol. 9, 2279-2283.
  5. Itoh, H. et al. (2002) Eur. J. Biochem. 269, 5931-5938.
  6. Gupta, S. and Knowlton, A.A. J. Cell Mol. Med. 9, 51-58.
  7. Deocaris, C.C. et al. (2006) Cell Stress Chaperones 11, 116-128.
  8. Lai, H.C. et al. (2007) Am. J. Physiol. Endocrinol. Metab. 292, E292-E297.
Entrez-Gene Id
3329
Swiss-Prot Acc.
P10809
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.

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Product # Size Price
56831S
100 µl (50 tests) $ 341.0

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