Human ATP13A2 ELISA Kit (HUEB2421)- High Sensitivity

Product Name:	Human Probable cation-transporting ATPase 13A2 (ATP13A2) ELISA Kit
SKU:	HUEB2421
Size:	96T
Target:	Human Probable cation-transporting ATPase 13A2 (ATP13A2)
Synonyms:	PARK9
Assay Type:	Competitive
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.78-50ng/mL
Sensitivity:	0.23ng/mL

Intra CV:

4.6%

Inter CV:

7.4%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	89-89%	106-116%	85-94%	99-108%
EDTA Plasma(N=5)	100-112%	91-101%	89-99%	99-108%
Heparin Plasma(N=5)	114-124%	81-91%	109-120%	100-110%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	88	82-94
Plasma	90	84-96

Function:

ATPase that plays a role in intracellular cation homeostasis and the maintenance of neuronal integrity (PubMed:22186024). Required for a proper lysosomal and mitochondrial maintenance (PubMed:22296644, PubMed:28137957).

Uniprot:	Q9NQ11
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Cation-transporting ATPase 13A2
Research Area:	Neurosciences
Subcellular Location:	Membrane Multi-pass membrane protein Lysosome Lysosome membrane
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	ATP13A2: May play a role in intracellular cation homeostasis and the maintenance of neuronal integrity. Expressed in brain; protein levels are markedly increased in brain from subjects with Parkinson disease and subjects with dementia with Lewy bodies. Detected in pyramidal neurons located throughout the cingulate cortex. In the substantia nigra, it is found in neuromelanin- positive dopaminergic neurons. Belongs to the cation transport ATPase (P-type) (TC 3.A.3) family. Type V subfamily. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Membrane protein, integral; Transporter, ion channel; EC 3.6.3.-; Transporter; Hydrolase; Membrane protein, multi-pass Chromosomal Location of Human Ortholog: 1p36 Cellular Component: autophagic vacuole; cell soma; integral to plasma membrane; late endosome; lysosomal lumen; lysosomal membrane; lysosome; multivesicular body; neuron projection; transport vesicle; vesicle Molecular Function:cation-transporting ATPase activity; manganese ion binding; protein binding; zinc ion binding Biological Process: cellular calcium ion homeostasis; cellular cation homeostasis; cellular iron ion homeostasis; cellular zinc ion homeostasis; positive regulation of protein secretion; protein amino acid autophosphorylation; regulation of autophagic vacuole size; regulation of endopeptidase activity; regulation of macroautophagy; zinc ion homeostasis Disease: Kufor-rakeb Syndrome
NCBI Summary:	This gene encodes a member of the P5 subfamily of ATPases which transports inorganic cations as well as other substrates. Mutations in this gene are associated with Kufor-Rakeb syndrome (KRS), also referred to as Parkinson disease 9. Multiple transcript variants encoding different isoforms have been found for this gene.[provided by RefSeq, Nov 2008]
UniProt Code:	Q9NQ11
NCBI GenInfo Identifier:	14285364
NCBI Gene ID:	23400
NCBI Accession:	Q9NQ11.2
UniProt Secondary Accession:	Q9NQ11,O75700, Q5JXY1, Q5JXY2, Q6S9Z9,
UniProt Related Accession:	Q9NQ11
Molecular Weight:	128,323 Da
NCBI Full Name:	Probable cation-transporting ATPase 13A2
NCBI Synonym Full Names:	ATPase 13A2
NCBI Official Symbol:	ATP13A2
NCBI Official Synonym Symbols:	CLN12; KRPPD; PARK9; HSA9947
NCBI Protein Information:	probable cation-transporting ATPase 13A2
UniProt Protein Name:	Probable cation-transporting ATPase 13A2
Protein Family:	Probable cation-transporting ATPase
UniProt Gene Name:	ATP13A2
UniProt Entry Name:	AT132_HUMAN

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	Protocol
1.	Add Sample: Add 50µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of the micro ELISA plate well, avoiding inside wall touching and foaming as much as possible.
2.	Detection Reagent A: Immediately add 50µL of Detection Reagent A working solution to each well. Cover with the Plate sealer and gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy, warm it to room temperature until the solution is uniform.
3.	Wash: Aspirate each well and wash three times. Wash by filling each well with Wash Buffer (approximately 400µL) and let it sit in the well for 1-2 minutes. Ensure complete removal of liquid at each step. After the last wash, invert the plate and pat it against thick, clean absorbent paper.
4.	Detection Reagent B: Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 45 minutes at 37°C.
5.	Wash: Repeat the wash process five times as conducted in step 3.
6.	Substrate: Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C, protecting the plate from light. Adjust the reaction time as needed, but do not exceed 30 minutes.
7.	Stop: Add 50µL of Stop Solution to each well. If the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	OD Measurement: Determine the optical density (OD value) of each well using a micro-plate reader set to 450 nm. Preheat the instrument and set the testing parameters in advance.
9.	After experiment: Store all reagents according to their specified storage temperatures until their expiry date.

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 ATP13A2 ELISA Kit