Porcine HSPA5 ELISA Kit (PREB0438)- High Sensitivity

Product Name:	Porcine 78 kDa glucose-regulated protein (HSPA5) ELISA Kit
SKU:	PREB0438
Size:	96T
Target:	Porcine 78 kDa glucose-regulated protein (HSPA5)
Synonyms:	78 kDa glucose-regulated protein, Binding-immunoglobulin protein, Heat shock protein 70 family protein 5, Heat shock protein family A member 5, Immunoglobulin heavy chain-binding protein, GRP-78, BiP, HSP70 family protein 5, GRP78
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Pig
Detection Range:	0.156-10ng/ml
Sensitivity:	0.064ng/ml

Intra CV:

Provided with the Kit

Inter CV:

Provided with the Kit

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	94-104%	111-111%	106-116%	110-122%
EDTA Plasma(N=5)	94-103%	101-114%	87-97%	82-92%
Heparin Plasma(N=5)	98-107%	99-111%	96-106%	99-110%

Recovery:

Provided with the Kit

Function:

Endoplasmic reticulum chaperone that plays a key role in protein folding and quality control in the endoplasmic reticulum lumen (By similarity). Involved in the correct folding of proteins and degradation of misfolded proteins via its interaction with DNAJC10/ERdj5, probably to facilitate the release of DNAJC10/ERdj5 from its substrate (By similarity). Acts as a key repressor of the ERN1/IRE1-mediated unfolded protein response (UPR). In the unstressed endoplasmic reticulum, recruited by DNAJB9/ERdj4 to the luminal region of ERN1/IRE1, leading to disrupt the dimerization of ERN1/IRE1, thereby inactivating ERN1/IRE1. Accumulation of misfolded protein in the endoplasmic reticulum causes release of HSPA5/BiP from ERN1/IRE1, allowing homodimerization and subsequent activation of ERN1/IRE1 (By similarity). Plays an auxiliary role in post-translational transport of small presecretory proteins across endoplasmic reticulum (ER). May function as an allosteric modulator for SEC61 channel-forming translocon complex, likely cooperating with SEC62 to enable the productive insertion of these precursors into SEC61 channel. Appears to specifically regulate translocation of precursors having inhibitory residues in their mature region that weaken channel gating.

Uniprot:	P34935
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant pig Endoplasmic reticulum chaperone BiP
Sub Unit:	Monomer and homooligomer; homooligomerization via the interdomain linker inactivates the chaperone activity and acts as a storage of HSPA5/BiP molecules (By similarity). Interacts with DNAJC1 (via J domain) (By similarity). Component of an EIF2 complex at least composed of CELF1/CUGBP1, CALR, CALR3, EIF2S1, EIF2S2, HSP90B1 and HSPA5 (By similarity). Part of a large chaperone multiprotein complex comprising DNAJB11, HSP90B1, HSPA5, HYOU, PDIA2, PDIA4, PDIA6, PPIB, SDF2L1, UGT1A1 and very small amounts of ERP29, but not, or at very low levels, CALR nor CANX (By similarity). Interacts with TMEM132A and TRIM21. May form a complex with ERLEC1, OS9, SEL1L and SYVN1. Interacts with DNAJC10 (By similarity). Interacts with DNAJB9/ERdj4; leading to recruit HSPA5/BiP to ERN1/IRE1. Interacts with ERN1/IRE1; interaction takes place following interaction with DNAJB9/ERdj4 and leads to inactivate ERN1/IRE1 (By similarity). Interacts with MX1 (By similarity). Interacts with METTL23. Interacts with CEMIP; the interaction induces calcium leakage from the endoplasmic reticulum and cell migration. Interacts with PCSK4 form; the interaction takes place in the endoplasmic reticulum. Interacts with CIPC. Interacts with CCDC88B (via C-terminus); the interaction opposes ERN1-mediated JNK activation, protecting against apoptosis. Interacts with INPP5K; necessary for INPP5K localization at the endoplasmic reticulum. Interacts with LOXL2; leading to activate the ERN1/IRE1-XBP1 pathway of the unfolded protein response (By similarity). Interacts with CLU under stressed condition; interaction increases CLU protein stability; facilitates its retrotranslocation and redistribution to the mitochondria; cooperatively suppress stress-induced apoptosis by stabilizing mitochondrial membrane integrity (By similarity). Interacts with CCDC47 (By similarity).
Research Area:	Neurosciences
Subcellular Location:	Endoplasmic reticulum lumen
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	Endoplasmic reticulum chaperone that plays a key role in protein folding and quality control in the endoplasmic reticulum lumen (). Involved in the correct folding of proteins and degradation of misfolded proteins via its interaction with DNAJC10/ERdj5, probably to facilitate the release of DNAJC10/ERdj5 from its substrate (). Acts as a key repressor of the ERN1/IRE1-mediated unfolded protein response (UPR). In the unstressed endoplasmic reticulum, recruited by DNAJB9/ERdj4 to the luminal region of ERN1/IRE1, leading to disrupt the dimerization of ERN1/IRE1, thereby inactivating ERN1/IRE1. Accumulation of misfolded protein in the endoplasmic reticulum causes release of HSPA5/BiP from ERN1/IRE1, allowing homodimerization and subsequent activation of ERN1/IRE1 ().
UniProt Protein Details:
NCBI Summary:
UniProt Code:	P34935
NCBI GenInfo Identifier:	14916993
NCBI Gene ID:	407060
NCBI Accession:	P34935.2
UniProt Secondary Accession:	P34935
UniProt Related Accession:	P34935
Molecular Weight:	28,912 Da
NCBI Full Name:	Endoplasmic reticulum chaperone BiP
NCBI Synonym Full Names:
NCBI Official Symbol:	HSPA5
NCBI Official Synonym Symbols:	BIP; GRP78
NCBI Protein Information:	78 kDa glucose-regulated protein
UniProt Protein Name:	Endoplasmic reticulum chaperone BiP
UniProt Synonym Protein Names:	78 kDa glucose-regulated protein
Protein Family:
UniProt Gene Name:	HSPA5
UniProt Entry Name:

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.