Rat HSPA5/GRP78/BiP ELISA Kit (RTFI00414)

Product Name:	Rat HSPA5/GRP78/BiP ELISA Kit
Product Code:	RTFI00414
Size:	96 Assays
Target:	Rat Hspa5
Alias:	Hspa5, GRP-78, Grp78, Steroidogenesis-activator polypeptide, Heat shock 70 kDa protein 5, Immunoglobulin heavy chain-binding protein, BiP
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat Hspa5 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Hspa5 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-105	98
EDTA plasma(n=5)	90-99	95
UFH plasma(n=5)	88-99	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Hspa5 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-102%	88-95%	85-103%
EDTA plasma(n=5)	91-97%	84-101%	83-101%
UFH plasma(n=5)	87-96%	82-92%	80-99%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P06761

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:Heat shock protein; Chaperone Cellular Component: cell surface; endoplasmic reticulum; endoplasmic reticulum lumen; endoplasmic reticulum membrane; ER-Golgi intermediate compartment; focal adhesion; integral to endoplasmic reticulum membrane; melanosome; membrane; midbody; mitochondrion; myelin sheath; nucleus; plasma membrane; signalosome; smooth endoplasmic reticulum Molecular Function:ATP binding; ATPase activity; enzyme binding; glycoprotein binding; misfolded protein binding; protein binding; protein domain specific binding; ribosome binding; ubiquitin protein ligase binding; unfolded protein binding Biological Process: cellular response to glucose starvation; cerebellar Purkinje cell layer development; cerebellum structural organization; ER overload response; negative regulation of apoptosis; negative regulation of transforming growth factor beta receptor signaling pathway; positive regulation of cell migration; positive regulation of protein ubiquitination; proteolysis involved in cellular protein catabolic process; substantia nigra development; unfolded protein response, activation of signaling protein activity
NCBI Summary:	secreted protein of the endoplasmic reticulum; may be involved in the assembly of secreted and membrane-bound proteins [RGD, Feb 2006]
UniProt Code:	P06761
NCBI GenInfo Identifier:	121574
NCBI Gene ID:	25617
NCBI Accession:	P06761.1
UniProt Related Accession:	P06761
Molecular Weight:	72,347 Da
NCBI Full Name:	78 kDa glucose-regulated protein
NCBI Synonym Full Names:	heat shock protein family A member 5
NCBI Official Symbol:	Hspa5
NCBI Official Synonym Symbols:	BIP; GRP78
NCBI Protein Information:	78 kDa glucose-regulated protein
UniProt Protein Name:	78 kDa glucose-regulated protein
UniProt Synonym Protein Names:	Heat shock 70 kDa protein 5; Immunoglobulin heavy chain-binding protein; BiP; Steroidogenesis-activator polypeptide
Protein Family:	KDEL motif-containing protein
UniProt Gene Name:	Hspa5
UniProt Entry Name:	GRP78_RAT

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37°C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.