Product Name:	Human anti-HSPA5 (anti-Heat shock protein family A member 5) ELISA Kit
Product Code:	HUFI08015
Size:	96 Assays
Alias:	anti-HSPA5 ELISA Kit
Detection method:	Indirect ELISA - Biotin
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human anti-HSPA5 (anti-Heat shock protein family A member 5) concentrations in serum plasma and other biological fluids.
Sensitivity:	< 0.234ng/ml
Range:	0.391-25ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Human anti-HSPA5 (anti-Heat shock protein family A member 5) and the recovery rates were calculated by comparing the measured value to the expected amount of Human anti-HSPA5 (anti-Heat shock protein family A member 5) in samples. Enquire for more information.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human anti-HSPA5 (anti-Heat shock protein family A member 5) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
CV(%):	Intra-Assay: CV<8% Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate(Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

UniProt Protein Function:	GRP78: a member of the HSP family of molecular chaperones required for endoplasmic reticulum integrity and stress-induced autophagy. Plays a central role in regulating the unfolded protein response (UPR), and is an obligatory component of autophagy in mammalian cells. May play an important role in cellular adaptation and oncogenic survival. One of the client proteins of GRP78 is protein double-stranded RNA-activated protein-like endoplasmic reticulum kinase (PERK). Probably plays a role in facilitating the assembly of multimeric protein complexes inside the ER.
UniProt Protein Details:	Protein type:Chaperone; Heat shock protein Chromosomal Location of Human Ortholog: 9q33.3 Cellular Component: signalosome; endoplasmic reticulum membrane; focal adhesion; smooth endoplasmic reticulum; cell surface; mitochondrion; endoplasmic reticulum; endoplasmic reticulum lumen; ER-Golgi intermediate compartment; membrane; melanosome; plasma membrane; integral to endoplasmic reticulum membrane; midbody; nucleus Molecular Function:protein domain specific binding; protein binding; enzyme binding; chaperone binding; ubiquitin protein ligase binding; unfolded protein binding; ATPase activity; ribosome binding; calcium ion binding; misfolded protein binding; glycoprotein binding; ATP binding Biological Process: platelet activation; ER-associated protein catabolic process; cerebellum structural organization; unfolded protein response; cerebellar Purkinje cell layer development; cellular response to glucose starvation; platelet degranulation; substantia nigra development; cellular protein metabolic process; unfolded protein response, activation of signaling protein activity; positive regulation of protein ubiquitination; blood coagulation; negative regulation of transforming growth factor beta receptor signaling pathway; ER overload response; positive regulation of cell migration; negative regulation of apoptosis
NCBI Summary:	The protein encoded by this gene is a member of the heat shock protein 70 (HSP70) family. It is localized in the lumen of the endoplasmic reticulum (ER), and is involved in the folding and assembly of proteins in the ER. As this protein interacts with many ER proteins, it may play a key role in monitoring protein transport through the cell.[provided by RefSeq, Sep 2010]
UniProt Code:	P11021
NCBI GenInfo Identifier:	14916999
NCBI Gene ID:	3309
NCBI Accession:	P11021.2
UniProt Secondary Accession:	P11021,Q2EF78, Q9NPF1, Q9UK02, B0QZ61,
UniProt Related Accession:	P11021
Molecular Weight:
NCBI Full Name:	78 kDa glucose-regulated protein
NCBI Synonym Full Names:	heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)
NCBI Official Symbol:	HSPA5
NCBI Official Synonym Symbols:	BIP; MIF2; GRP78; HEL-S-89n
NCBI Protein Information:	78 kDa glucose-regulated protein; immunoglobulin heavy chain-binding protein; epididymis secretory sperm binding protein Li 89n; endoplasmic reticulum lumenal Ca(2+)-binding protein grp78
UniProt Protein Name:	78 kDa glucose-regulated protein
UniProt Synonym Protein Names:	Endoplasmic reticulum lumenal Ca(2+)-binding protein grp78; Heat shock 70 kDa protein 5; Immunoglobulin heavy chain-binding protein; BiP
UniProt Gene Name:	HSPA5
UniProt Entry Name:	GRP78_HUMAN

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

Before adding to the wells, equilibrate the TMB substrate for at least 30 mins at 37 ? ?C. When diluting samples and reagents, ensure they are mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample (diluted at least « with Sample Dilution Buffer) and control (blank) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (blank) wells!
2.	Aliquot 100 µL of standard solutions into the standard wells.
3.	Add 100 µL of properly diluted Sample into the sample wells.
4.	Seal the plate with a cover and incubate at 37 °C for 90 mins.
5.	Wash: Repeat the aspiration/wash process for 2 times.
6.	Add 100 µL of Biotin-labelled antibody working solution into the bottom of each well (standard, test sample & zero wells) without touching the side walls.
7.	Seal the plate with a cover and incubate at 37 °C for 60 mins.
8.	Wash: Remove the cover, and wash plate 3 times with Wash buffer.
9.	HRP-Streptavidin Conjugate (SABC): Add 100?l of SABC Working Solution into each well, cover the plate and incubate at 37 °C for 30 minutes.
10.	Wash: Remove the cover and wash plate 5 times with Wash Buffer.
11.	TMB Substrate: Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37 °C in dark for 10-20 mins. (Note: This incubation time is for reference only, the optimal time should be determined by the end-user.) As soon as a blue colour develops in the first 3-4 wells (with most concentrated standards) and the other wells show no obvious colour, terminate the reaction.
12.	Add 50 µL of Stop solution into each well and mix thoroughly. The colour changes into yellow immediately.
13.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.