Rat Atp2a1 ELISA Kit (RTFI00399)- High Sensitivity

Product Name:	Rat Atp2a1 (Sarcoplasmic/endoplasmic reticulum calcium ATPase 1) ELISA Kit
Product Code:	RTFI00399
Size:	96 Assays
Target:	Rat Atp2a1
Alias:	Atp2a1, SERCA1, SR Ca, 2+-ATPase 1, Sarcoplasmic, endoplasmic reticulum calcium ATPase 1, Endoplasmic reticulum class 1, 2 Ca, 2+ ATPase, Calcium pump 1, Calcium-transporting ATPase sarcoplasmic reticulum type, fast twitch skeletal muscle isoform
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 Atp2a1 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Atp2a1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-95	91
EDTA plasma(n=5)	85-105	97
UFH plasma(n=5)	87-104	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Atp2a1 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)	86-104%	87-95%	88-103%
EDTA plasma(n=5)	83-101%	86-100%	85-99%
UFH plasma(n=5)	84-100%	89-100%	83-95%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

Q64578

UniProt Protein Function:	ATP2A1: This magnesium-dependent enzyme catalyzes the hydrolysis of ATP coupled with the translocation of calcium from the cytosol to the sarcoplasmic reticulum lumen. Contributes to calcium sequestration involved in muscular excitation/contraction. Associated with sarcolipin (SLN) and phospholamban (PLN). Increased contractile activity leads to a decrease in SERCA1 expression, while decreased contractile activity leads to an increase in SERCA1 expression. Skeletal muscle, fast twitch muscle (type II) fibers. Reversibly inhibited by phospholamban (PLN) at low calcium concentrations. Dephosphorylated PLN decreases the apparent affinity of the ATPase for calcium. This inhibition is regulated by the phosphorylation of PLN. Belongs to the cation transport ATPase (P-type) (TC 3.A.3) family. Type IIA subfamily. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 3.6.3.8; Transporter, ion channel; Endoplasmic reticulum; Hydrolase; Membrane protein, multi-pass; Transporter; Membrane protein, integral Chromosomal Location of Human Ortholog: 16p12.1 Cellular Component: I band; endoplasmic reticulum membrane; sarcoplasmic reticulum membrane; membrane; sarcoplasmic reticulum; perinuclear region of cytoplasm; integral to membrane; ER-Golgi intermediate compartment Molecular Function:protein binding; protein homodimerization activity; calcium-transporting ATPase activity; calcium ion binding; ATP binding Biological Process: apoptotic mitochondrial changes; positive regulation of fast-twitch skeletal muscle contraction; maintenance of mitochondrion localization; metabolic process; reduction of endoplasmic reticulum calcium ion concentration; elevation of endoplasmic reticulum calcium ion concentration; calcium ion transport; regulation of striated muscle contraction; blood coagulation; transmembrane transport; elevation of mitochondrial calcium ion concentration; negative regulation of striated muscle contraction Disease: Brody Myopathy
NCBI Summary:	This gene encodes one of the SERCA Ca(2+)-ATPases, which are intracellular pumps located in the sarcoplasmic or endoplasmic reticula of muscle cells. This enzyme catalyzes the hydrolysis of ATP coupled with the translocation of calcium from the cytosol to the sarcoplasmic reticulum lumen, and is involved in muscular excitation and contraction. Mutations in this gene cause some autosomal recessive forms of Brody disease, characterized by increasing impairment of muscular relaxation during exercise. Alternative splicing results in three transcript variants encoding different isoforms. [provided by RefSeq, Oct 2013]
UniProt Code:	Q64578
NCBI GenInfo Identifier:	554506571
NCBI Gene ID:	487
NCBI Accession:	NP_001273004.1
UniProt Secondary Accession:	Q64578,O14984, A8K5J9, B3KY17,
UniProt Related Accession:	O14983
Molecular Weight:	95,199 Da
NCBI Full Name:	sarcoplasmic/endoplasmic reticulum calcium ATPase 1 isoform c
NCBI Synonym Full Names:	ATPase, Ca++ transporting, cardiac muscle, fast twitch 1
NCBI Official Symbol:	ATP2A1
NCBI Official Synonym Symbols:	ATP2A; SERCA1
NCBI Protein Information:	sarcoplasmic/endoplasmic reticulum calcium ATPase 1; calcium pump 1; SR Ca(2+)-ATPase 1; endoplasmic reticulum class 1/2 Ca(2+) ATPase; calcium-transporting ATPase sarcoplasmic reticulum type, fast twitch skeletal muscle isoform
UniProt Protein Name:	Sarcoplasmic/endoplasmic reticulum calcium ATPase 1
UniProt Synonym Protein Names:	Calcium pump 1; Calcium-transporting ATPase sarcoplasmic reticulum type, fast twitch skeletal muscle isoform; Endoplasmic reticulum class 1/2 Ca(2+) ATPase
Protein Family:	Sarcoplasmic/endoplasmic reticulum calcium ATPase
UniProt Gene Name:	ATP2A1
UniProt Entry Name:	AT2A1_HUMAN

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.