Human PCNA ELISA Kit (HUES03268)- High Sensitivity

Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.31-20 ng/mL
Sensitivity:	0.19 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human PCNA in samples. No significant cross-reactivity or interference between Human PCNA and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human PCNA. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human PCNA and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human PCNA, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human PCNA. The concentration of Human PCNA in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	PCNA: This protein is an auxiliary protein of DNA polymerase delta and is involved in the control of eukaryotic DNA replication by increasing the polymerase's processibility during elongation of the leading strand. Induces a robust stimulatory effect on the 3'- 5' exonuclease and 3'-phosphodiesterase, but not apurinic- apyrimidinic (AP) endonuclease, APEX2 activities. Has to be loaded onto DNA in order to be able to stimulate APEX2. Homotrimer. Forms a complex with activator 1 heteropentamer in the presence of ATP. Interacts with EXO1, POLH, POLK, DNMT1, ERCC5, FEN1, CDC6 and POLDIP2. Interacts with APEX2; this interaction is triggered by reactive oxygen species and increased by misincorporation of uracil in nuclear DNA. Forms a ternary complex with DNTTIP2 and core histone. Interacts with KCTD10 and PPP1R15A. Interacts with POLD1, POLD3 and POLD4. Interacts with BAZ1B; the interaction is direct. Interacts with HLTF and SHPRH. Interacts with NUDT15. Interaction is disrupted in response to UV irradiation and acetylation. Interacts with p21Cip1/p21(CIP1) and CDT1; interacts via their PIP-box which also recruits the DCX(DTL) complex. Interacts with DDX11. Interacts with EGFR; positively regulates PCNA. Interacts with C12orf48/PARI. Interacts with SMARCAD1. Belongs to the PCNA family.
UniProt Protein Details:	Protein type:DNA replication; Cell cycle regulation Chromosomal Location of Human Ortholog: 20pter-p12 Cellular Component: nucleoplasm; centrosome; PCNA complex; nuclear replication fork; cytoplasm; DNA replication factor C complex; nucleus Molecular Function:identical protein binding; protein binding; DNA polymerase processivity factor activity; MutLalpha complex binding; purine-specific mismatch base pair DNA N-glycosylase activity; receptor tyrosine kinase binding; dinucleotide insertion or deletion binding Biological Process: mismatch repair; heart development; positive regulation of deoxyribonuclease activity; DNA strand elongation during DNA replication; response to lipid; DNA repair; leading strand elongation; G1/S-specific transcription in mitotic cell cycle; telomere maintenance via semi-conservative replication; cell proliferation; bypass DNA synthesis; response to cadmium ion; epithelial cell differentiation; base-excision repair; nucleotide-excision repair; transcription-coupled nucleotide-excision repair; telomere maintenance via recombination; nucleotide-excision repair, DNA gap filling; mitotic cell cycle; telomere maintenance; regulation of DNA replication; G1/S transition of mitotic cell cycle Disease: Ataxia-telangiectasia-like Disorder 2
NCBI Summary:	The protein encoded by this gene is found in the nucleus and is a cofactor of DNA polymerase delta. The encoded protein acts as a homotrimer and helps increase the processivity of leading strand synthesis during DNA replication. In response to DNA damage, this protein is ubiquitinated and is involved in the RAD6-dependent DNA repair pathway. Two transcript variants encoding the same protein have been found for this gene. Pseudogenes of this gene have been described on chromosome 4 and on the X chromosome. [provided by RefSeq, Jul 2008]
UniProt Code:	P12004
NCBI GenInfo Identifier:	129694
NCBI Gene ID:	5111
NCBI Accession:	P12004. 1
UniProt Secondary Accession:	P12004,B2R897, D3DW02,
UniProt Related Accession:	P12004
Molecular Weight:	261
NCBI Full Name:	Proliferating cell nuclear antigen
NCBI Synonym Full Names:	proliferating cell nuclear antigen
NCBI Official Symbol:	PCNA
NCBI Official Synonym Symbols:	ATLD2
NCBI Protein Information:	proliferating cell nuclear antigen; cyclin; DNA polymerase delta auxiliary protein
UniProt Protein Name:	Proliferating cell nuclear antigen
UniProt Synonym Protein Names:	Cyclin
Protein Family:	PCNA-associated factor
UniProt Gene Name:	PCNA
UniProt Entry Name:	PCNA_HUMAN

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (ng/mL)	O.D	Average	Corrected
20	2.359 2.411	2.385	2.301
10	1.605 1.607	1.606	1.522
5	1.022 1.012	1.017	0.933
2.5	0.536 0.552	0.544	0.46
1.25	0.271 0.257	0.264	0.18
0.63	0.191 0.185	0.188	0.104
0.31	0.128 0.144	0.136	0.052
0	0.075 0.093	0.084	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human PCNA were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human PCNA were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	1.10	2.10	7.82	1.09	2.24	7.74
Standard deviation	0.07	0.09	0.28	0.06	0.13	0.31
C V (%)	6.36	4.29	3.58	5.50	5.80	4.01

Recovery

The recovery of Human PCNA spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	94-106	100
EDTA plasma (n=5)	84-99	91
Cell culture media (n=5)	84-96	91

Linearity

Samples were spiked with high concentrations of Human PCNA and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	93-108	95-106	90-102
1:2	Average (%)	101	101	95
1:4	Range (%)	88-101	86-98	86-100
1:4	Average (%)	95	92	92
1:8	Range (%)	92-102	87-101	84-96
1:8	Average (%)	97	92	89
1:16	Range (%)	88-99	80-93	82-93
1:16	Average (%)	94	86	88

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro ELISA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.