Product Name:	Human LPCAT2 ELISA Kit
Product Code:	HUFI01832
Size:	96 Assays
Alias:	LPCAT2, LPC acyltransferase 2, LPCAT-2, LysoPC acyltransferase 2, Lysophosphatidic acid acyltransferase alpha, LPAAT-alpha, Acetyl-CoA, lyso-PAF acetyltransferase
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human LPCAT2 concentrations in serum plasma and other biological fluids.
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 Human LPCAT2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human LPCAT2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-104	95
EDTA plasma(n=5)	87-105	97
UFH plasma(n=5)	87-104	98

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human LPCAT2 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)	93-103%	86-99%	89-103%
EDTA plasma(n=5)	82-96%	84-99%	86-99%
UFH plasma(n=5)	81-100%	85-98%	80-97%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

Q7L5N7

UniProt Protein Function:	AYTL1: Possesses both acyltransferase and acetyltransferase activities. Activity is calcium-dependent. Involved in platelet- activating factor (PAF) biosynthesis by catalyzing the conversion of the PAF precursor, 1-O-alkyl-sn-glycero-3-phosphocholine (lyso- PAF) into 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF). Also converts lyso-PAF to 1-alkyl-phosphatidylcholine (PC), a major component of cell membranes and a PAF precursor. Under resting conditions, acyltransferase activity is preferred. Upon acute inflammatory stimulus, acetyltransferase activity is enhanced and PAF synthesis increases. Belongs to the 1-acyl-sn-glycerol-3-phosphate acyltransferase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 2.3.1.23; EC 2.3.1.67; Acetyltransferase; Membrane protein, integral; EC 2.3.1.51 Chromosomal Location of Human Ortholog: 16q12.2 Cellular Component: Golgi membrane; endoplasmic reticulum membrane; Golgi stack; endoplasmic reticulum; integral to membrane; lipid particle Molecular Function:1-alkylglycerophosphocholine O-acetyltransferase activity; 1-acylglycerophosphocholine O-acyltransferase activity; calcium ion binding; 1-acylglycerol-3-phosphate O-acyltransferase activity Biological Process: platelet activating factor biosynthetic process; phospholipid metabolic process; glycerophospholipid biosynthetic process
NCBI Summary:	This gene encodes a member of the lysophospholipid acyltransferase family. The encoded enzyme may function in two ways: to catalyze the biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) from 1-O-alkyl-sn-glycero-3-phosphocholine, and to catalyze the synthesis of glycerophospholipid precursors from arachidonyl-CoA and lysophosphatidylcholine. The encoded protein may function in membrane biogenesis and production of platelet-activating factor in inflammatory cells. The enzyme may localize to the endoplasmic reticulum and the Golgi. [provided by RefSeq, Feb 2009]
UniProt Code:	Q7L5N7
NCBI GenInfo Identifier:	74738601
NCBI Gene ID:	54947
NCBI Accession:	Q7L5N7.1
UniProt Secondary Accession:	Q7L5N7,Q6MZJ6, Q9NX23, A3KBM1,
UniProt Related Accession:	Q7L5N7
Molecular Weight:	544
NCBI Full Name:	Lysophosphatidylcholine acyltransferase 2
NCBI Synonym Full Names:	lysophosphatidylcholine acyltransferase 2
NCBI Official Symbol:	LPCAT2
NCBI Official Synonym Symbols:	AYTL1; LysoPAFAT
NCBI Protein Information:	lysophosphatidylcholine acyltransferase 2; LPCAT-2; 1-AGPAT 11; LPAAT-alpha; LPC acyltransferase 2; acyltransferase like 1; acyltransferase-like 1; 1-AGP acyltransferase 11; lysoPC acyltransferase 2; lyso-PAF acetyltransferase; acetyl-CoA:lyso-PAF acetyltransferase; lysophosphatidic acid acyltransferase alpha; 1-acylglycerophosphocholine O-acyltransferase; 1-acylglycerol-3-phosphate O-acyltransferase 11; 1-alkylglycerophosphocholine O-acetyltransferase; acyl-CoA:lysophosphatidylcholine acyltransferase 2; lyso-platelet-activating factor (PAF) acetyltransferase; acetyl-CoA:lyso-platelet-activating factor acetyltransferase
UniProt Protein Name:	Lysophosphatidylcholine acyltransferase 2
UniProt Synonym Protein Names:	1-acylglycerol-3-phosphate O-acyltransferase 11 (EC:2.3.1.51); 1-AGP acyltransferase 11; 1-AGPAT 11; 1-acylglycerophosphocholine O-acyltransferase; 1-alkylglycerophosphocholine O-acetyltransferase (EC:2.3.1.67); Acetyl-CoA:lyso-platelet-activating factor acetyltransferase; Acetyl-CoA:lyso-PAF acetyltransferase; Lyso-PAF acetyltransferase; LysoPAFAT; Acyltransferase-like 1; Lysophosphatidic acid acyltransferase alpha; LPAAT-alpha
Protein Family:	Lysophosphatidylcholine acyltransferase
UniProt Gene Name:	LPCAT2
UniProt Entry Name:	PCAT2_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
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 Âµl 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 Âµl 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 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.

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Human LPCAT2 ELISA Kit

Description