Product Name:	Human PSMA6 (Proteasome subunit alpha type-6)ELISA Kit
Product Code:	HUFI07012
Size:	96 Assays
Alias:	Proteasome subunit alpha type-6 ELISA Kit, 27 kDa prosomal protein ELISA Kit, PROS-27 ELISA Kit, p27K ELISA Kit, Macropain iota chain ELISA Kit, Multicatalytic endopeptidase complex iota chain ELISA Kit, Proteasome iota chain ELISA Kit, PROS27 ELISA Kit, PSMA6 ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PSMA6 (Proteasome subunit alpha type-6)ELISA Kit concentrations in serum plasma and other biological fluids.
Sensitivity:	< 18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human PSMA6 (Proteasome subunit alpha type-6)ELISA Kit and the recovery rates were calculated by comparing the measured value to the expected amount of Human PSMA6 (Proteasome subunit alpha type-6)ELISA Kit in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-104	99
EDTA plasma(n=5)	94-102	98
heparin plasma(n=5)	88-104	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PSMA6 (Proteasome subunit alpha type-6)ELISA Kit 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)	85-105%	87-102%	89-102%
EDTA plasma(n=5)	85-99%	82-98%	82-92%
UFH plasma(n=5)	80-99%	84-96%	80-94%

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 Protein Function:	PSMA6: The proteasome is a multicatalytic proteinase complex which is characterized by its ability to cleave peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH. The proteasome has an ATP-dependent proteolytic activity. The 26S proteasome consists of a 20S proteasome core and two 19S regulatory subunits. The 20S proteasome core is composed of 28 subunits that are arranged in four stacked rings, resulting in a barrel-shaped structure. The two end rings are each formed by seven alpha subunits, and the two central rings are each formed by seven beta subunits. The catalytic chamber with the active sites is on the inside of the barrel. Belongs to the peptidase T1A family.
UniProt Protein Details:	Protein type:Proteasome complex; EC 3.4.25.1; Protease Chromosomal Location of Human Ortholog: 14q13 Cellular Component: proteasome complex; nucleoplasm; polysome; proteasome core complex; sarcomere; nuclear matrix; myofibril; cytoplasm; nucleus; cytosol Molecular Function:threonine endopeptidase activity; NF-kappaB binding; protein binding; RNA binding; endopeptidase activity Biological Process: positive regulation of ubiquitin-protein ligase activity during mitotic cell cycle; negative regulation of ubiquitin-protein ligase activity during mitotic cell cycle; protein polyubiquitination; viral reproduction; apoptosis; antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; activation of NF-kappaB transcription factor; regulation of apoptosis; antigen processing and presentation of peptide antigen via MHC class I; proteolysis involved in cellular protein catabolic process; regulation of ubiquitin-protein ligase activity during mitotic cell cycle; anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process; regulation of inflammatory response; antigen processing and presentation of exogenous peptide antigen via MHC class I; gene expression; mitotic cell cycle; regulation of amino acid metabolic process; G1/S transition of mitotic cell cycle; negative regulation of apoptosis Disease: Myocardial Infarction, Susceptibility To
NCBI Summary:	The proteasome is a multicatalytic proteinase complex with a highly ordered ring-shaped 20S core structure. The core structure is composed of 4 rings of 28 non-identical subunits; 2 rings are composed of 7 alpha subunits and 2 rings are composed of 7 beta subunits. Proteasomes are distributed throughout eukaryotic cells at a high concentration and cleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway. An essential function of a modified proteasome, the immunoproteasome, is the processing of class I MHC peptides. This gene encodes a member of the peptidase T1A family, that is a 20S core alpha subunit. Multiple transcript variants encoding several different isoforms have been found for this gene. A pseudogene has been identified on the Y chromosome. [provided by RefSeq, Aug 2013]
UniProt Code:	P60900
NCBI GenInfo Identifier:	46397659
NCBI Gene ID:	5687
NCBI Accession:	P60900.1
UniProt Secondary Accession:	P60900,P34062, Q6IB60, B2R7J9, B4DQR4, B4DXJ9,
UniProt Related Accession:	P60900
Molecular Weight:	246
NCBI Full Name:	Proteasome subunit alpha type-6
NCBI Synonym Full Names:	proteasome (prosome, macropain) subunit, alpha type, 6
NCBI Official Symbol:	PSMA6
NCBI Official Synonym Symbols:	IOTA; p27K; PROS27
NCBI Protein Information:	proteasome subunit alpha type-6; PROS-27; macropain iota chain; prosomal P27K protein; proteasome iota chain; macropain subunit iota; 27 kDa prosomal protein; proteasome subunit iota; multicatalytic endopeptidase complex iota chain
UniProt Protein Name:	Proteasome subunit alpha type-6
UniProt Synonym Protein Names:	27 kDa prosomal protein; PROS-27; p27K; Macropain iota chain; Multicatalytic endopeptidase complex iota chain; Proteasome iota chain
Protein Family:	Proteasome
UniProt Gene Name:	PSMA6
UniProt Entry Name:	PSA6_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.