Rat CHRM2/Muscarinic Acetylcholine Receptor M2 ELISA Kit (RTFI00664)

Product Name:	Rat CHRM2/Muscarinic Acetylcholine Receptor M2 ELISA Kit
Product Code:	RTFI00664
Size:	96 Assays
Target:	Rat CHRM2
Alias:	CHRM2, HM2, mAChR M2, 7TM receptor, cholinergic receptor, muscarinic 2, muscarinic acetylcholine receptor M2, muscarinic M2 receptor
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	37.5 pmol/ml
Range:	62.5-4000pmol/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat CHRM2 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat CHRM2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-103	94
EDTA plasma(n=5)	85-101	91
UFH plasma(n=5)	94-104	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat CHRM2 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%	94-105%	86-105%
EDTA plasma(n=5)	82-96%	84-101%	86-100%
UFH plasma(n=5)	81-99%	80-100%	82-97%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P10980

UniProt Protein Function:	mAChR M2: The muscarinic acetylcholine receptor mediates various cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides and modulation of potassium channels through the action of G proteins. Primary transducing effect is adenylate cyclase inhibition. Genetic variations in CHRM2 can influence susceptibility to major depressive disorder (MDD). MDD is one of the most common psychiatric disorders. MDD is a complex trait characterized by one or more major depressive episodes without a history of manic, mixed, or hypomanic episodes. A major depressive episode is characterized by at least 2 weeks during which there is a new onset or clear worsening of either depressed mood or loss of interest or pleasure in nearly all activities. Four additional symptoms must also be present including changes in appetite, weight, sleep, and psychomotor activity; decreased energy; feelings of worthlessness or guilt; difficulty thinking, concentrating, or making decisions; or recurrent thoughts of death or suicidal ideation, plans, or attempts. The episode must be accompanied by distress or impairment in social, occupational, or other important areas of functioning. Belongs to the G-protein coupled receptor 1 family. Muscarinic acetylcholine receptor subfamily. CHRM2 sub-subfamily.
UniProt Protein Details:	Protein type:Membrane protein, integral; Receptor, GPCR; Membrane protein, multi-pass; GPCR, family 1 Cellular Component: asymmetric synapse; postsynaptic membrane; membrane; cell soma; integral to plasma membrane; dendrite; plasma membrane; nerve terminal; cell junction Molecular Function:drug binding; G-protein coupled acetylcholine receptor activity Biological Process: regulation of smooth muscle contraction; synaptic transmission; elevation of cytosolic calcium ion concentration during G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); acetylcholine receptor signaling, muscarinic pathway; response to virus; regulation of heart contraction
NCBI Summary:	may inhibit cholinergic-mediated bronchoconstriction; may regulate bladder contraction [RGD, Feb 2006]
UniProt Code:	P10980
NCBI GenInfo Identifier:	13591922
NCBI Gene ID:	81645
NCBI Accession:	NP_112278.1
UniProt Secondary Accession:	P10980,Q9Z2Z1,
UniProt Related Accession:	P10980
Molecular Weight:	51,539 Da
NCBI Full Name:	muscarinic acetylcholine receptor M2
NCBI Synonym Full Names:	cholinergic receptor, muscarinic 2
NCBI Official Symbol:	Chrm2
NCBI Official Synonym Symbols:	Acm2
NCBI Protein Information:	muscarinic acetylcholine receptor M2; 7TM receptor; muscarinic receptor m2; M2 muscarinic acetylcholine receptor; cholinergic receptor, muscarinic 2, cardiac
UniProt Protein Name:	Muscarinic acetylcholine receptor M2
Protein Family:	Muscarinic acetylcholine receptor
UniProt Gene Name:	Chrm2
UniProt Entry Name:	ACM2_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.