Rat Connexin 40/GJA5 ELISA Kit (RTFI00708)

Product Name:	Rat Connexin 40/GJA5 ELISA Kit
Product Code:	RTFI00708
Size:	96 Assays
Target:	Rat CX40
Alias:	CX40, ATFB11, GJA5, alpha 5, 40kD, connexin 40, connexin-40, Connexin-40, Cx40, gap junction alpha-5 protein, gap junction protein, alpha 5, 40kDa, gap junction protein, alpha 5, 40kDa, connexin 40, MGC11185
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	23.438pg/ml
Range:	39.063-2500pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat CX40 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat CX40 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-102	92
EDTA plasma(n=5)	89-98	92
UFH plasma(n=5)	88-101	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat CX40 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)	85-104%	87-104%	85-103%
EDTA plasma(n=5)	84-100%	84-101%	84-100%
UFH plasma(n=5)	84-95%	81-99%	93-97%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P28234

UniProt Protein Function:	GJA5: One gap junction consists of a cluster of closely packed pairs of transmembrane channels, the connexons, through which materials of low MW diffuse from one cell to a neighboring cell. Defects in GJA5 are a cause of familial atrial standstill (FAS). Atrial standstill is an extremely rare arrhythmia, characterized by the absence of electrical and mechanical activity in the atria. Electrocardiographically, it is characterized by bradycardia, the absence of P waves, and a junctional narrow complex escape rhythm. Defects in GJA5 are the cause of familial atrial fibrillation type 11 (ATFB11). ATFB11 is a familial form of atrial fibrillation, a common sustained cardiac rhythm disturbance. Atrial fibrillation is characterized by disorganized atrial electrical activity and ineffective atrial contraction promoting blood stasis in the atria and reduces ventricular filling. It can result in palpitations, syncope, thromboembolic stroke, and congestive heart failure. Belongs to the connexin family. Alpha-type (group II) subfamily.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Channel, misc.; Membrane protein, integral; Membrane protein, multi-pass Cellular Component: connexon complex; cell projection; integral to plasma membrane; gap junction Molecular Function:gap junction hemi-channel activity; gap junction channel activity Biological Process: blood vessel development; negative regulation of glomerular filtration; gap junction assembly; regulation of vasodilation; heart development; positive regulation of vasodilation; protein oligomerization; positive regulation of vasoconstriction; regulation of systemic arterial blood pressure; regulation of systemic arterial blood pressure by renal system process; artery morphogenesis; embryonic heart tube development; negative regulation of blood pressure; angiogenesis; transmembrane transport; skeletal development; potassium ion transport; embryonic limb morphogenesis
NCBI Summary:	forms intercellular connoxin channels in gap junctions; mediates cell-cell communication [RGD, Feb 2006]
UniProt Code:	P28234
NCBI GenInfo Identifier:	9506725
NCBI Gene ID:	50563
NCBI Accession:	NP_062153.1
UniProt Related Accession:	P28234
Molecular Weight:
NCBI Full Name:	gap junction alpha-5 protein
NCBI Synonym Full Names:	gap junction protein, alpha 5
NCBI Official Symbol:	Gja5
NCBI Official Synonym Symbols:	Cx40
NCBI Protein Information:	gap junction alpha-5 protein; connexin 40; connexin-40; gap junction membrane channel protein alpha 5
UniProt Protein Name:	Gap junction alpha-5 protein
UniProt Synonym Protein Names:	Connexin-40
UniProt Gene Name:	Gja5
UniProt Entry Name:	CXA5_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.