Human PARP1 ELISA Kit (HUFI00837)

Product Name:	Human PARP1 ELISA Kit
Product Code:	HUFI00837
Size:	96 Assays
Alias:	PARP1, ADPRT, PPOL, Poly [ADP-ribose] polymerase 1, PARP-1, Poly[ADP-ribose] synthase 1, ADPRT 1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PARP1 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.375ng/ml
Range:	0.625-40ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human PARP1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human PARP1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	92-101	97
EDTA plasma(n=5)	86-104	95
UFH plasma(n=5)	87-98	91

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PARP1 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)	89-97%	86-94%	88-104%
EDTA plasma(n=5)	84-85%	84-101%	84-99%
UFH plasma(n=5)	80-90%	80-94%	86-96%

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

P09874

UniProt Protein Function:	PARP1: Involved in the base excision repair (BER) pathway, by catalyzing the poly(ADP-ribosyl)ation of a limited number of acceptor proteins involved in chromatin architecture and in DNA metabolism. This modification follows DNA damages and appears as an obligatory step in a detection/signaling pathway leading to the reparation of DNA strand breaks. Mediates the poly(ADP- ribosyl)ation of APLF and CHFR. Positively regulates the transcription of MTUS1 and negatively regulates the transcription of MTUS2/TIP150. With EEF1A1 and TXK, forms a complex that acts as a T-helper 1 (Th1) cell-specific transcription factor and binds the promoter of IFN-gamma to directly regulate its transcription, and is thus involved importantly in Th1 cytokine production. Component of a base excision repair (BER) complex, containing at least XRCC1, PARP2, POLB and LRIG3. Homo- and heterodimer with PARP2. Interacts with PARP3, APTX and SRY. The SWAP complex consists of NPM1, NCL, PARP1 and SWAP70. Interacts with TIAM2 and ZNF423. Interacts (when poly-ADP- ribosylated) with CHD1L. Interacts with the DNA polymerase alpha catalytic subunit POLA1; this interaction functions as part of the control of replication fork progression. Interacts with EEF1A1, RNF4 and TXK.
UniProt Protein Details:	Protein type:DNA repair, damage; EC 2.4.2.30; Nuclear envelope; Nuclear receptor co-regulator; Nucleolus; Transferase Chromosomal Location of Human Ortholog: 1q42.12 Cellular Component: membrane; mitochondrion; nuclear chromosome, telomeric region; nuclear envelope; nucleolus; nucleoplasm; nucleus; protein complex; transcription factor complex Molecular Function:DNA ligase (ATP) activity; enzyme binding; identical protein binding; NAD+ ADP-ribosyltransferase activity; protein binding; protein kinase binding; protein N-terminus binding; RNA binding; transcription factor binding Biological Process: cellular response to insulin stimulus; DNA ligation during DNA repair; DNA repair; double-strand break repair; double-strand break repair via homologous recombination; lagging strand elongation; macrophage differentiation; mitochondrial DNA metabolic process; mitochondrial DNA repair; mitochondrion organization and biogenesis; negative regulation of transcription from RNA polymerase II promoter; nucleotide-excision repair, DNA damage recognition; nucleotide-excision repair, DNA duplex unwinding; nucleotide-excision repair, DNA incision; nucleotide-excision repair, DNA incision, 3'-to lesion; nucleotide-excision repair, DNA incision, 5'-to lesion; nucleotide-excision repair, preincision complex assembly; nucleotide-excision repair, preincision complex stabilization; peptidyl-serine ADP-ribosylation; protein amino acid ADP-ribosylation; regulation of catalytic activity; response to DNA damage stimulus; telomere maintenance; transcription from RNA polymerase II promoter
NCBI Summary:	This gene encodes a chromatin-associated enzyme, poly(ADP-ribosyl)transferase, which modifies various nuclear proteins by poly(ADP-ribosyl)ation. The modification is dependent on DNA and is involved in the regulation of various important cellular processes such as differentiation, proliferation, and tumor transformation and also in the regulation of the molecular events involved in the recovery of cell from DNA damage. In addition, this enzyme may be the site of mutation in Fanconi anemia, and may participate in the pathophysiology of type I diabetes. [provided by RefSeq, Jul 2008]
UniProt Code:	P09874
NCBI GenInfo Identifier:	130781
NCBI Gene ID:	142
NCBI Accession:	P09874.4
UniProt Secondary Accession:	P09874,Q8IUZ9, B1ANJ4,
UniProt Related Accession:	P09874
Molecular Weight:	113kDa
NCBI Full Name:	Poly
NCBI Synonym Full Names:	poly(ADP-ribose) polymerase 1
NCBI Official Symbol:	PARP1
NCBI Official Synonym Symbols:	PARP; PPOL; ADPRT; ARTD1; ADPRT1; PARP-1; ADPRT 1; pADPRT-1
NCBI Protein Information:	poly [ADP-ribose] polymerase 1
UniProt Protein Name:	Poly [ADP-ribose] polymerase 1
UniProt Synonym Protein Names:	ADP-ribosyltransferase diphtheria toxin-like 1; ARTD1; NAD(+) ADP-ribosyltransferase 1; ADPRT 1; Poly[ADP-ribose] synthase 1
Protein Family:	Poly [ADP-ribose] polymerase
UniProt Gene Name:	PARP1

*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.