Product Name:	Human PADI4 / PAD4 ELISA Kit
Product Code:	HUFI02693
Size:	96 Assays
Alias:	PADI4, PADPAD4, PDI4, PDI5EC 3.5.3.15, peptidyl arginine deiminase, type V, Peptidylarginine deiminase IV, Protein-arginine deiminase type IV, protein-arginine deiminase type-4
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PADI4 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.113ng/ml
Range:	0.188-12ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human PADI4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human PADI4 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-105	98
EDTA plasma(n=5)	86-93	90
UFH plasma(n=5)	87-105	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PADI4 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)	91-97%	90-104%	85-92%
EDTA plasma(n=5)	83-100%	85-99%	83-97%
UFH plasma(n=5)	85-96%	81-95%	83-99%

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

Q9UM07

UniProt Protein Function:	PADI4: Catalyzes the citrullination/deimination of arginine residues of proteins. Citrullinates histone H3 at 'Arg-8' and/or 'Arg-17' and histone H4 at 'Arg-3', which prevents their methylation by CARM1 and HRMT1L2/PRMT1 and represses transcription. Citrullinates EP300/P300 at 'Arg-2142', which favors its interaction with NCOA2/GRIP1. Genetic variations in PADI4 are a cause of susceptibility to rheumatoid arthritis (RA). It is a systemic inflammatory disease with autoimmune features and a complex genetic component. It primarily affects the joints and is characterized by inflammatory changes in the synovial membranes and articular structures, widespread fibrinoid degeneration of the collagen fibers in mesenchymal tissues, and by atrophy and rarefaction of bony structures. Could have an important role in the pathogenesis of rheumatoid arthritis by increasing citrullination of proteins in rheumatoid arthritis synovial tissues, leading, in a cytokine-rich milieu, to a break in tolerance to citrullinated peptides processed and presented in the appropriate HLA context. Belongs to the protein arginine deiminase family.
UniProt Protein Details:	Protein type:Nuclear receptor co-regulator; Hydrolase; EC 3.5.3.15 Chromosomal Location of Human Ortholog: 1p36.13 Cellular Component: cytoplasm; nucleus Molecular Function:arginine deiminase activity; protein binding; calcium ion binding; protein-arginine deiminase activity Biological Process: chromatin remodeling; nucleosome assembly; transcription, DNA-dependent; regulation of transcription, DNA-dependent; stem cell maintenance; innate immune response; protein modification process; chromatin modification; peptidyl-citrulline biosynthetic process from peptidyl-arginine Disease: Rheumatoid Arthritis
NCBI Summary:	This gene is a member of a gene family which encodes enzymes responsible for the conversion of arginine residues to citrulline residues. This gene may play a role in granulocyte and macrophage development leading to inflammation and immune response. [provided by RefSeq, Jul 2008]
UniProt Code:	Q9UM07
NCBI GenInfo Identifier:	296439260
NCBI Gene ID:	23569
NCBI Accession:	Q9UM07.2
UniProt Related Accession:	Q9UM07
Molecular Weight:
NCBI Full Name:	Protein-arginine deiminase type-4
NCBI Synonym Full Names:	peptidyl arginine deiminase 4
NCBI Official Symbol:	PADI4
NCBI Official Synonym Symbols:	PAD; PAD4; PDI4; PDI5; PADI5
NCBI Protein Information:	protein-arginine deiminase type-4
UniProt Protein Name:	Protein-arginine deiminase type-4
UniProt Synonym Protein Names:	HL-60 PAD; Peptidylarginine deiminase IV; Protein-arginine deiminase type IV
Protein Family:	Lipase
UniProt Gene Name:	PADI4
UniProt Entry Name:	PADI4_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.

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Human PADI4 / PAD4 ELISA Kit

Description