Product Name:	Human PYCARD (Apoptosis-associated speck-like protein containing a CARD) ELISA Kit
Product Code:	HUFI08097
Size:	96 Assays
Alias:	hASC ELISA Kit, Caspase recruitment domain-containing protein 5 ELISA Kit, PYD and CARD domain-containing protein ELISA Kit, Target of methylation-induced silencing 1 ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PYCARD (Apoptosis-associated speck-like protein containing a CARD) concentrations in serum plasma and other biological fluids.
Sensitivity:	< 46.875pg/ml
Range:	78.125-5000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human PYCARD (Apoptosis-associated speck-like protein containing a CARD) and the recovery rates were calculated by comparing the measured value to the expected amount of Human PYCARD (Apoptosis-associated speck-like protein containing a CARD) in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	93-105	99
EDTA plasma(n=5)	89-101	95
heparin plasma(n=5)	88-104	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PYCARD (Apoptosis-associated speck-like protein containing a CARD) 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)	93-98%	86-105%	87-103%
EDTA plasma(n=5)	84-100%	88-98%	86-96%
UFH plasma(n=5)	85-87%	81-98%	80-91%

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:	PYCARD: Promotes caspase-mediated apoptosis. This proapoptotic activity is mediated predominantly through the activation of caspase-9. May be a component of the inflammasome, a protein complex which also includes NALP2, CARD8 and CASP1 and whose function would be the activation of proinflammatory caspases. Forms complexes with other DAPIN domain-containing proteins. Interacts with CIAS1/PYPAF1 and PYDC1. Widely expressed at low levels. Detected in peripheral blood leukocytes, lung, small intestine, spleen, thymus, colon and at lower levels in placenta, liver and kidney. Very low expression in skeletal muscle, heart and brain. Detected in the leukemia cell lines HL-60 and U-937, but not in Jurkat T- cell lymphoma and Daudi Burkitt's lymphoma. Detected in the melanoma cell line WM35, but not in WM793. Not detected in HeLa cervical carcinoma cells and MOLT-4 lymphocytic leukemia cells. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Tumor suppressor; Adaptor/scaffold Chromosomal Location of Human Ortholog: 16p11.2 Cellular Component: cell soma; mitochondrion; endoplasmic reticulum; cytoplasm; nucleolus; extracellular region; IkappaB kinase complex; cytosol; nucleus Molecular Function:protein binding; protein homodimerization activity; protease binding; caspase activator activity; Pyrin domain binding Biological Process: myeloid dendritic cell activation during immune response; apoptosis; positive regulation of apoptosis; regulation of protein stability; positive regulation of caspase activity; positive regulation of JNK cascade; positive regulation of interleukin-1 beta secretion; positive regulation of activated T cell proliferation; signal transduction; defense response to Gram-negative bacterium; activation of NF-kappaB transcription factor; tumor necrosis factor-mediated signaling pathway; positive regulation of phagocytosis; inflammatory response; DNA damage response, signal transduction by p53 class mediator resulting in induction of apoptosis; defense response to virus; positive regulation of adaptive immune response; caspase activation; interleukin-1 beta production; positive regulation of interleukin-6 production; negative regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of tumor necrosis factor production; negative regulation of interferon-beta production; myeloid dendritic cell activation; positive regulation of interferon-gamma production; positive regulation of actin filament polymerization; inhibition of NF-kappaB transcription factor; regulation of inflammatory response; innate immune response; regulation of autophagy; positive regulation of transcription factor activity; positive regulation of T cell activation; positive regulation of defense response to virus by host; activation of innate immune response; positive regulation of antigen processing and presentation of peptide antigen via MHC class II
NCBI Summary:	This gene encodes an adaptor protein that is composed of two protein-protein interaction domains: a N-terminal PYRIN-PAAD-DAPIN domain (PYD) and a C-terminal caspase-recruitment domain (CARD). The PYD and CARD domains are members of the six-helix bundle death domain-fold superfamily that mediates assembly of large signaling complexes in the inflammatory and apoptotic signaling pathways via the activation of caspase. In normal cells, this protein is localized to the cytoplasm; however, in cells undergoing apoptosis, it forms ball-like aggregates near the nuclear periphery. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	Q9ULZ3
NCBI GenInfo Identifier:	18203507
NCBI Gene ID:	29108
NCBI Accession:	Q9ULZ3.2
UniProt Related Accession:	Q9ULZ3
Molecular Weight:	21 KD
NCBI Full Name:	Apoptosis-associated speck-like protein containing a CARD
NCBI Synonym Full Names:	PYD and CARD domain containing
NCBI Official Symbol:	PYCARD
NCBI Official Synonym Symbols:	ASC; TMS; TMS1; CARD5; TMS-1
NCBI Protein Information:	apoptosis-associated speck-like protein containing a CARD
UniProt Protein Name:	Apoptosis-associated speck-like protein containing a CARD
UniProt Synonym Protein Names:	Caspase recruitment domain-containing protein 5; PYD and CARD domain-containing protein; Target of methylation-induced silencing 1
Protein Family:	Ascalin
UniProt Gene Name:	PYCARD
UniProt Entry Name:	ASC_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.