Human HSPD1 ELISA Kit (HUEB0869)- High Sensitivity

Product Name:	Human 60 kDa heat shock protein, mitochondrial (HSPD1) ELISA Kit
SKU:	HUEB0869
Size:	96T
Target:	Human 60 kDa heat shock protein, mitochondrial (HSPD1)
Synonyms:	60 kDa chaperonin, Chaperonin 60, Heat shock protein 60, HuCHA60, Mitochondrial matrix protein P1, P60 lymphocyte protein, CPN60, HSP-60, HSP60
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.045ng/mL

Intra CV:

4.3%

Inter CV:

7.5%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	103-116%	103-112%	90-100%	98-109%
EDTA Plasma(N=5)	111-120%	81-81%	108-118%	80-89%
Heparin Plasma(N=5)	91-101%	105-114%	92-104%	97-107%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	96	90-102
Plasma	98	92-104

Function:

Chaperonin implicated in mitochondrial protein import and macromolecular assembly. Together with Hsp10, facilitates the correct folding of imported proteins. May also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix (PubMed:1346131, PubMed:11422376). The functional units of these chaperonins consist of heptameric rings of the large subunit Hsp60, which function as a back-to-back double ring. In a cyclic reaction, Hsp60 ring complexes bind one unfolded substrate protein per ring, followed by the binding of ATP and association with 2 heptameric rings of the co-chaperonin Hsp10. This leads to sequestration of the substrate protein in the inner cavity of Hsp60 where, for a certain period of time, it can fold undisturbed by other cell components. Synchronous hydrolysis of ATP in all Hsp60 subunits results in the dissociation of the chaperonin rings and the release of ADP and the folded substrate protein.

Uniprot:	P10809
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human 60 kDa heat shock protein, mitochondrial
Sub Unit:	(Microbial infection) Interacts with HBV protein X and HTLV-1 protein p40tax (PubMed:15120623, PubMed:1731090).
Research Area:	Epigenetics
Subcellular Location:	Mitochondrion matrix
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	HSP60: Implicated in mitochondrial protein import and macromolecular assembly. May facilitate the correct folding of imported proteins. May also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix. Interacts with HRAS. Interacts with HBV protein X and HTLV-1 protein p40tax. Belongs to the chaperonin (HSP60) family.
UniProt Protein Details:	Protein type:Chaperone; Mitochondrial Chromosomal Location of Human Ortholog: 2q33.1 Cellular Component: extracellular space; cell surface; protein complex; mitochondrion; early endosome; coated pit; cytosol; secretory granule; membrane; mitochondrial matrix; cytoplasm; mitochondrial inner membrane; plasma membrane; lipopolysaccharide receptor complex; cyclin-dependent protein kinase activating kinase holoenzyme complex Molecular Function:protein binding; p53 binding; lipopolysaccharide binding; ubiquitin protein ligase binding; chaperone binding; ATPase activity; double-stranded RNA binding; unfolded protein binding; DNA replication origin binding; single-stranded DNA binding; ATP binding Biological Process: B cell proliferation; caspase activation; B cell activation; T cell activation; protein stabilization; viral reproduction; positive regulation of apoptosis; positive regulation of interleukin-12 production; protein maturation; isotype switching to IgG isotypes; positive regulation of interleukin-6 production; B cell cytokine production; positive regulation of interleukin-10 production; response to unfolded protein; MyD88-dependent toll-like receptor signaling pathway; positive regulation of interferon-gamma production; 'de novo' protein folding; protein refolding; positive regulation of T cell activation; positive regulation of T cell mediated immune response to tumor cell; chaperone-mediated protein complex assembly; positive regulation of interferon-alpha production; negative regulation of apoptosis; positive regulation of macrophage activation Disease: Spastic Paraplegia 13, Autosomal Dominant; Leukodystrophy, Hypomyelinating, 4
NCBI Summary:	This gene encodes a member of the chaperonin family. The encoded mitochondrial protein may function as a signaling molecule in the innate immune system. This protein is essential for the folding and assembly of newly imported proteins in the mitochondria. This gene is adjacent to a related family member and the region between the 2 genes functions as a bidirectional promoter. Several pseudogenes have been associated with this gene. Two transcript variants encoding the same protein have been identified for this gene. Mutations associated with this gene cause autosomal recessive spastic paraplegia 13. [provided by RefSeq, Jun 2010]
UniProt Code:	P10809
NCBI GenInfo Identifier:	129379
NCBI Gene ID:	3329
NCBI Accession:	P10809.2
UniProt Secondary Accession:	P10809,Q38L19, Q9UCR6, B2R5M6, B7Z712,
UniProt Related Accession:	P10809
Molecular Weight:	17,100 Da
NCBI Full Name:	60 kDa heat shock protein, mitochondrial
NCBI Synonym Full Names:	heat shock 60kDa protein 1 (chaperonin)
NCBI Official Symbol:	HSPD1
NCBI Official Synonym Symbols:	HLD4; CPN60; GROEL; HSP60; HSP65; SPG13; HSP-60; HuCHA60
NCBI Protein Information:	60 kDa heat shock protein, mitochondrial; chaperonin 60; 60 kDa chaperonin; heat shock protein 65; P60 lymphocyte protein; mitochondrial matrix protein P1; short heat shock protein 60 Hsp60s1
UniProt Protein Name:	60 kDa heat shock protein, mitochondrial
UniProt Synonym Protein Names:	60 kDa chaperonin; Chaperonin 60; CPN60; Heat shock protein 60; HSP-60; Hsp60; HuCHA60; Mitochondrial matrix protein P1; P60 lymphocyte protein
Protein Family:	60 kDa heat shock protein
UniProt Gene Name:	HSPD1
UniProt Entry Name:	CH60_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

ELISA	Antibodies
Human HSP60 ELISA Kit	Anti-Hsp60 Antibody (CAB0564)
Human HSP-60/HSPD1 (Heat Shock Protein 60) CLIA Kit (HUES01029)	Anti-Hsp60 Antibody (CAB0969)
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	HSPB1 Antibody (PACO07002)
	HSPBP1 Antibody (PACO09834)