Human ATG12 ELISA Kit (HUEB2355)- High Sensitivity

Product Name:	Human Ubiquitin-like protein ATG12 (ATG12) ELISA Kit
SKU:	HUEB2355
Size:	96T
Target:	Human Ubiquitin-like protein ATG12 (ATG12)
Synonyms:	Autophagy-related protein 12, APG12-like, APG12, APG12L
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.078ng/mL

Intra CV:

7.1%

Inter CV:

9.7%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	97-107%	88-97%	103-115%	102-112%
EDTA Plasma(N=5)	105-117%	92-104%	98-107%	80-90%
Heparin Plasma(N=5)	106-116%	110-122%	112-120%	101-112%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	104	98-110
Plasma	106	100-112

Function:

Ubiquitin-like protein involved in autophagy vesicles formation. Conjugation with ATG5 through a ubiquitin-like conjugating system involving also ATG7 as an E1-like activating enzyme and ATG10 as an E2-like conjugating enzyme, is essential for its function. The ATG12-ATG5 conjugate acts as an E3-like enzyme which is required for lipidation of ATG8 family proteins and their association to the vesicle membranes. The ATG12-ATG5 conjugate also regulates negatively the innate antiviral immune response by blocking the type I IFN production pathway through direct association with RARRES3 and MAVS. Plays also a role in translation or delivery of incoming viral RNA to the translation apparatus.

Uniprot:	O94817
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Ubiquitin-like protein ATG12
Sub Unit:	Forms a conjugate with ATG5. The ATG12-ATG5 conjugate forms a complex with several units of ATG16L. Interacts with ATG3, ATG7 and ATG10. ATG12-ATG5 also interacts with MAVS, MGA, RARRES3 and TECPR1.
Research Area:	Cancer
Subcellular Location:	Cytoplasm Preautophagosomal structure membrane Peripheral membrane protein TECPR1 recruits the ATG12-ATG5 conjugate to the autolysosomal membrane.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	ATG12: Ubiquitin-like protein required for autophagy. Conjugated to ATG3 and ATG5. Ubiquitous. Belongs to the ATG12 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Autophagy; Ubiquitin-like modifier Chromosomal Location of Human Ortholog: 5q22.3 Cellular Component: cytosol; phagocytic vesicle membrane; pre-autophagosomal structure membrane Molecular Function:APG8 conjugating enzyme activity; protein binding Biological Process: autophagic vacuole formation; C-terminal protein lipidation; macroautophagy; mitochondrion degradation
NCBI Summary:	Autophagy is a process of bulk protein degradation in which cytoplasmic components, including organelles, are enclosed in double-membrane structures called autophagosomes and delivered to lysosomes or vacuoles for degradation. ATG12 is the human homolog of a yeast protein involved in autophagy (Mizushima et al., 1998 [PubMed 9852036]).[supplied by OMIM, Mar 2008]
UniProt Code:	O94817
NCBI GenInfo Identifier:	17366241
NCBI Gene ID:	9140
NCBI Accession:	O94817.1
UniProt Secondary Accession:	O94817,Q6PJV2,
UniProt Related Accession:	O94817
Molecular Weight:	15kDa
NCBI Full Name:	Ubiquitin-like protein ATG12
NCBI Synonym Full Names:	autophagy related 12
NCBI Official Symbol:	ATG12
NCBI Official Synonym Symbols:	APG12; FBR93; APG12L; HAPG12
NCBI Protein Information:	ubiquitin-like protein ATG12
UniProt Protein Name:	Ubiquitin-like protein ATG12
UniProt Synonym Protein Names:	Autophagy-related protein 12; APG12-like
Protein Family:	Autophagy protein
UniProt Gene Name:	ATG12

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA

Human ATG12 / APG12 ELISA Kit