Description
Heat shock proteins (Hsp) are a set of highly conserved proteins that include constitutively expressed (Hsp60, Hsp70, and Hsp90) and stress-induced (Hsp27 and Hsp72) proteins. Hsp60 is localized to the mitochondria, where it promotes mitochondrial protein folding and facilitates proteolytic degradation of misfolded or denatured proteins. It binds Hsp10, which regulates the substrate binding and ATPase activity of Hsp60. In HeLa and Jurkat mitochondria, Hsp60 associates with caspase-3 to form a complex that dissociates and releases from the mitochondria during apoptosis. In addition, Hsp60 accelerates the maturation of procaspase-3 through its ATP-dependent "foldase" activity. In addition to its role in protein folding, Hsp60 has also been implicated in immune function. In macrophages, its binding to the toll-like receptor-4 complex induces production of TNFα and nitric oxide and stimulation of a proinflammatory response. Thus, the protein folding function of Hsp60 is involved in mitochondrial protein folding in both normal and apoptotic cells, while release of Hsp60 during necrosis is thought to stimulate a proinflammatory response.
This antibody is routinely tested by immunofluorescent imaging. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.
Format
- FormatFITC
- Excitation SourceBlue 488 nm
- Excitation Max494nm
- Emission Max520nm
FITC, fluorescein isothiocyanate, is a fluorochrome with a molecular weight of 389 Da. FITC is sensitive to pH changes and photobleaching. Due to nearly identical excitation and emission properties but different spillover characteristics, FITC and Alexa Fluor® 488 cannot be used simultaneously. FITC is relatively dim and should be reserved for highly expressed markers whenever possible.
Other Formats→
- Alexa Fluor® 647
- Purified
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Preparation and Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.Store undiluted at -20°C.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
Methanol Procedure for a 96 well plate:
Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 90% methanol. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Wash three times with PBS. Flick out PBS and add second step reagent. Incubate for 1 hour at RT. Wash three times with PBS. Image sample.
Triton-X 100 Procedure for a 96 well plate:
Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 0.1% Triton-X 100. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Flick out and wash three times with PBS. Flick out and add second step reagent. Incubate for 1 hour at RT. Flick out and wash three times with PBS. Image sample.
