Human MAP1LC3A ELISA Kit (HUFI01900)

Product Name:	Human MAP1LC3A ELISA Kit
Product Code:	HUFI01900
Size:	96 Assays
Alias:	MAP1LC3A, Apg8, APG8a, Apg8p3, ATG8E, MAP1ALC3, MLP3A, ATG8E, Autophagy-related protein LC3 A, Autophagy-related ubiquitin-like modifier LC3 A, LC3, LC3A, lc3-i, ii, MAP1A, 1B light chain 3 A, MAP1ALC3MAP1A, MAP1B LC3 A, MAP1BLC3MAP1A, MAP1B light chain 3 A, microtubule-associated protein 1 light chain 3 alphaMAP1 light chain 3-like protein 1, microtubule-associated proteins 1A, 1B light chain 3, microtubule-associated proteins 1A, 1B light chain 3A
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human MAP1LC3A concentrations in serum plasma and other biological fluids.
Sensitivity:	4.688pg/ml
Range:	7.813-500pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human MAP1LC3A and the recovery rates were calculated by comparing the measured value to the expected amount of Human MAP1LC3A in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	90-105	96
EDTA plasma(n=5)	85-105	96
UFH plasma(n=5)	85-105	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human MAP1LC3A 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)	85-103%	89-104%	89-105%
EDTA plasma(n=5)	84-100%	82-99%	86-96%
UFH plasma(n=5)	80-91%	85-95%	82-94%

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

Q9H492

UniProt Protein Function:	LC3A: is a ubiquitin-like protein that is a constituent of the ATG8-conjugation system, one of two evolutionarily conserved phosphatidylethanolamine conjugation systems necessary for the formation of the autophagosome. The human ATG8 system includes seven ubiquitin-like light chain proteins (LCPs) that are homologs of yeast LC3: MAP1LC3A, -B, -C, GABARAP, GABARAPL1, -2, and -3. Pro-LCPs are cleaved by ATG4B to expose a C-terminal glycine residue, the cytosolic LCP-I form. The exposed C-terminus is conjugated to the head group amine of phosphatidylethanolamine (PE) through an amide bond by a sequence of ubiquitination-like reactions that involves an E1 (ATG7), an E2 (ATG3), and an E3 (a complex including ATG5, ATG12, and ATG16L). The PE-congugated form (LCP-II) is tightly associated with the autophagosomal membrane. The LCP-II forms can also be delipidated by the ATG4 proteases: most of the LCPs are delipidated and liberated from the membrane before autophagosomes fuse with lysosomes. Two isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Microtubule-binding; Ubiquitin-like modifier; Autophagy; Vesicle Chromosomal Location of Human Ortholog: 20q11.22 Cellular Component: microtubule; extrinsic to membrane; late endosome; autophagic vacuole; cytoplasmic vesicle; cytosol Molecular Function:protein binding; phosphatidylethanolamine binding; microtubule binding; GABA receptor binding; phospholipid binding Biological Process: mitochondrion degradation; cellular response to nitrogen starvation; autophagic vacuole formation
NCBI Summary:	MAP1A and MAP1B are microtubule-associated proteins which mediate the physical interactions between microtubules and components of the cytoskeleton. MAP1A and MAP1B each consist of a heavy chain subunit and multiple light chain subunits. The protein encoded by this gene is one of the light chain subunits and can associate with either MAP1A or MAP1B. Two transcript variants encoding different isoforms have been found for this gene. The expression of variant 1 is suppressed in many tumor cell lines, suggesting that may be involved in carcinogenesis. [provided by RefSeq, Feb 2012]
UniProt Code:	Q9H492
NCBI GenInfo Identifier:	85701362
NCBI Gene ID:	84557
NCBI Accession:	Q9H492.2
UniProt Secondary Accession:	Q9H492,Q9GZQ8,
UniProt Related Accession:	Q9H492
Molecular Weight:
NCBI Full Name:	Microtubule-associated proteins 1A/1B light chain 3A
NCBI Synonym Full Names:	microtubule associated protein 1 light chain 3 alpha
NCBI Official Symbol:	MAP1LC3A
NCBI Official Synonym Symbols:	LC3; LC3A; ATG8E; MAP1ALC3; MAP1BLC3
NCBI Protein Information:	microtubule-associated proteins 1A/1B light chain 3A
UniProt Protein Name:	Microtubule-associated proteins 1A/1B light chain 3A
UniProt Synonym Protein Names:	Autophagy-related protein LC3 A; Autophagy-related ubiquitin-like modifier LC3 A; MAP1 light chain 3-like protein 1; MAP1A/MAP1B light chain 3 A; MAP1A/MAP1B LC3 A; Microtubule-associated protein 1 light chain 3 alpha
UniProt Gene Name:	MAP1LC3A
UniProt Entry Name:	MLP3A_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 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 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.