Rat Calpain-3/CAPN3 ELISA Kit (RTFI00496)- High Sensitivity

Product Name:	Rat Capn3 (Calpain-3) ELISA Kit
Product Code:	RTFI00496
Size:	96 Assays
Target:	Rat Capn3
Alias:	Capn3
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	0.375ng/ml
Range:	0.625-40ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat Capn3 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Capn3 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	93-105	98
EDTA plasma(n=5)	88-103	94
UFH plasma(n=5)	88-101	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Capn3 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)	86-105%	86-103%	86-102%
EDTA plasma(n=5)	85-96%	84-98%	83-87%
UFH plasma(n=5)	86-98%	80-94%	80-97%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P16259

UniProt Protein Function:	CAPN3: Calcium-regulated non-lysosomal thiol-protease. Defects in CAPN3 are the cause of limb-girdle muscular dystrophy type 2A (LGMD2A). LGMD2A is an autosomal recessive degenerative myopathy characterized by progressive symmetrical atrophy and weakness of the proximal limb muscles and elevated serum creatine kinase. The symptoms usually begin during the first two decades of life, and the disease gradually worsens, often resulting in loss of walking ability 10 or 20 years after onset. Belongs to the peptidase C2 family. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Calcium-binding; Cell development/differentiation; EC 3.4.22.54; Motility/polarity/chemotaxis; Protease Chromosomal Location of Human Ortholog: 3q35 Cellular Component: cytoplasm; cytosol; myofibril; nucleus; plasma membrane; protein complex; T-tubule; Z disc Molecular Function:calcium ion binding; calcium-dependent cysteine-type endopeptidase activity; catalytic activity; enzyme binding; ligase regulator activity; peptidase activity; protein binding; protein complex scaffold; receptor binding; sodium ion binding; structural constituent of muscle; titin binding Biological Process: activation of NF-kappaB transcription factor; myofibril assembly; negative regulation of apoptosis; negative regulation of protein sumoylation; negative regulation of transcription, DNA-dependent; positive regulation of proteolysis; positive regulation of release of sequestered calcium ion into cytosol; positive regulation of satellite cell activation involved in skeletal muscle regeneration; positive regulation of transcription, DNA-dependent; programmed cell death; protein complex assembly; proteolysis; regulation of catalytic activity; regulation of I-kappaB kinase/NF-kappaB cascade; regulation of myoblast differentiation; response to calcium ion; response to drug; response to muscle activity; sarcomere organization
NCBI Summary:	calcium-activated protease; alternative splicing produces different protein products; may be involved in astrocyte plasticity and in eye development and pathology [RGD, Feb 2006]
UniProt Code:	P16259
NCBI GenInfo Identifier:	8393041
NCBI Gene ID:	29155
NCBI Accession:	NP_058813.1
UniProt Related Accession:	P16259
Molecular Weight:	94,127 Da
NCBI Full Name:	calpain-3
NCBI Synonym Full Names:	calpain 3
NCBI Official Symbol:	Capn3
NCBI Official Synonym Symbols:	p94; Lp82; Lp84; Lp85
NCBI Protein Information:	calpain-3
UniProt Protein Name:	Calpain-3
UniProt Synonym Protein Names:	Calcium-activated neutral proteinase 3; CANP 3; Calpain L3; Calpain p94; Muscle-specific calcium-activated neutral protease 3
Protein Family:	Calpain
UniProt Gene Name:	Capn3

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.