Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Rat
Detection Method:	Colormetric
Detection Range:	0.16-10 ng/mL
Sensitivity:	0.10 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Rat PCNA in samples. No significant cross-reactivity or interference between Rat 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 Rat 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 Rat 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 Rat 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 Rat PCNA. The concentration of Rat 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 Cellular Component: cell; centrosome; cyclin-dependent protein kinase holoenzyme complex; cytoplasm; intracellular; nuclear chromosome, telomeric region; nuclear lamina; nuclear replication fork; nucleoplasm; nucleus; PCNA complex; replication fork Molecular Function:chromatin binding; damaged DNA binding; dinucleotide insertion or deletion binding; DNA polymerase processivity factor activity; enzyme binding; estrogen receptor binding; histone acetyltransferase binding; identical protein binding; MutLalpha complex binding; protein binding; purine-specific mismatch base pair DNA N-glycosylase activity; receptor tyrosine kinase binding; transcription factor binding Biological Process: base-excision repair, gap-filling; bypass DNA synthesis; epithelial cell differentiation; heart development; leading strand elongation; mismatch repair; negative regulation of transcription from RNA polymerase II promoter; positive regulation of deoxyribonuclease activity; positive regulation of DNA repair; positive regulation of DNA replication; replication fork processing; response to cadmium ion; response to estradiol stimulus; response to lipid; response to oxidative stress
NCBI Summary:	cell cycle protein that may contribute to the repair of DNA damage and to cell proliferation caused by exposure to pulmonary toxicants; may influence cell survival in ischaemically compromised tissue [RGD, Feb 2006]
UniProt Code:	P04961
NCBI GenInfo Identifier:	129698
NCBI Gene ID:	25737
NCBI Accession:	P04961. 1
UniProt Related Accession:	P04961
Molecular Weight:	28,749 Da
NCBI Full Name:	Proliferating cell nuclear antigen
NCBI Synonym Full Names:	proliferating cell nuclear antigen
NCBI Official Symbol:	Pcna
NCBI Official Synonym Symbols:	PCNAR; Pcna/cyclin
NCBI Protein Information:	proliferating cell nuclear antigen
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_RAT

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
10	2.308 2.314	2.311	2.248
5	1.613 1.627	1.62	1.557
2.5	0.884 0.846	0.865	0.802
1.25	0.403 0.441	0.422	0.359
0.63	0.227 0.203	0.215	0.152
0.32	0.161 0.157	0.159	0.096
0.16	0.111 0.115	0.113	0.05
0	0.054 0.072	0.063	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat 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)	0.48	1.55	3.80	0.48	1.61	4.12
Standard deviation	0.03	0.07	0.12	0.03	0.10	0.17
C V (%)	6.25	4.52	3.16	6.25	6.21	4.13

Recovery

The recovery of Rat 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)	89-105	96
EDTA plasma (n=5)	93-108	99
Cell culture media (n=5)	83-97	90

Linearity

Samples were spiked with high concentrations of Rat 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 (%)	94-110	85-100	93-105
1:2	Average (%)	100	91	99
1:4	Range (%)	93-107	80-94	83-93
1:4	Average (%)	99	86	88
1:8	Range (%)	86-101	84-97	88-102
1:8	Average (%)	93	91	94
1:16	Range (%)	91-105	86-100	87-99
1:16	Average (%)	98	93	92

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.