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QIA59 PCNA ELISA Kit

PCNA ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Detection Methods: Colorimetric
QIA59
  
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Overview

Replacement Information

Key Spec Table

Detection Methods
Colorimetric
Description
OverviewFast, sensitive assay for the in vitro quantitation of PCNA protein. The kit has broad reactivity, and is suitable to detect PCNA in autoimmune sera samples. The assay was validated using the BrdU Cell Proliferation Assay (Cat. No. QIA58).

This product has been discontinued.
Proliferating cell nuclear antigen (PCNA) has no known enzymatic activity; however, it can interact with a variety of proteins and regulate their activities. These varied interactions with DNA metabolic proteins imply that PCNA is a central factor for the coordination of DNA replication, DNA repair, epigenetic inheritance, and cell-cycle control.
Catalogue NumberQIA59
Brand Family Calbiochem®
Application Data
The mean signal of each standard run in replicates of two in four assays using two different batches of plates and two different lots of detector antibody. Note: 44 units/ml of the old standard (lysate) corresponds to about 200 ng/ml of the new standard.
Materials Required but Not Delivered• 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips.
• Automated plate washer, wash bottle or multichannel dispenser for washing.
• 1 liter graduated cylinder.
• Deionized or distilled H2O.
• 0.2 µm syringe filter and syringe.
• Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/595 nm or 450/540 nm. A single wavelength of 450 nm can also be used.
• Cell Resuspension Buffer: 50 mM Tris, containing 5 mM EDTA, 0.2 mM PMSF, 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin adjusted to pH 8.0.
References
ReferencesKelman, Z. and J. Hurwitz, 1998. Trends Biochem. Sci. 23, 236.
Montecucco, A., et al. 1998. The EMBO Journal 17, 3786.
Oku, T., et al. 1998. Genes Cells 3, 357.
Waga, S. and B. Stillman, 1998. Annu. Rev. Biochem. 67, 721.
Jonsson ZO and H. U, 1997. Bioessays 19, 967.
Prosperi, E., 1997. Prog. Cell Cycle Res. 3, 19.
Gulbis, J., et al. 1996. Cell, 87, 297.
Brott, D.A., et al. 1993. J. Cell. Biochem. 52, 362.
Product Information
Unit of DefinitionOne unit (U) is defined as the amount of PCNA measured from ~ 2.6 x 10³ SW620 cells.
Detection methodColorimetric
Form96 Tests
Format96-well strip plate
Kit contains96-Well Removable Strip Plate, 2 vials of PCNA Standard, Detector Antibody, 400X Conjugate, Conjugate Diluent, Substrate, Sample Diluent, 20X Plate Wash Concentrate, Antigen Extraction Agent, Stop Solution, Plate Sealers, and a user protocol.
Applications
Biological Information
Assay range3.125-200 ng/ml of PCNA
Assay time3.5 h
Sample TypeCell lysates, tissue culture media , serum, or plasma
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
R PhraseR: 23/24/25-35-36/37/38-40-43

Toxic by inhalation, in contact with skin and if swallowed.
Causes severe burns.
Irritating to eyes, respiratory system and skin.
Limited evidence of a carcinogenic effect.
May cause sensitization by skin contact.
S PhraseS: 26-36/37/39-45

In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
Wear suitable protective clothing, gloves and eye/face protection.
In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Product Usage Statements
Intended useThe Calbiochem® brand PCNA ELISA is a non-isotopic immunoassay for the in vitro quantitation of human, rat and mouse PCNA protein in cell lysates, tissue culture medium, sera and plasma.
Storage and Shipping Information
Ship Code Dry Ice Only
Toxicity Multiple Toxicity Values, refer to MSDS
Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Storage -20°C
Storage ConditionsUpon receipt of the kit, standards must be stored at -20°C. All other components may be stored at 4°C. Do not expose reagents to excessive light. Let kit sit at room temperature for 30 min before use. Note: This kit contains an overnight step during sample preparation.
Do not freeze Yes
Packaging Information
Transport Information
Supplemental Information
Kit contains96-Well Removable Strip Plate, 2 vials of PCNA Standard, Detector Antibody, 400X Conjugate, Conjugate Diluent, Substrate, Sample Diluent, 20X Plate Wash Concentrate, Antigen Extraction Agent, Stop Solution, Plate Sealers, and a user protocol.
Specifications
Global Trade Item Number
Catalogue Number GTIN
QIA59 0

Documentation

PCNA ELISA Kit Certificates of Analysis

TitleLot Number
QIA59

References

Reference overview
Kelman, Z. and J. Hurwitz, 1998. Trends Biochem. Sci. 23, 236.
Montecucco, A., et al. 1998. The EMBO Journal 17, 3786.
Oku, T., et al. 1998. Genes Cells 3, 357.
Waga, S. and B. Stillman, 1998. Annu. Rev. Biochem. 67, 721.
Jonsson ZO and H. U, 1997. Bioessays 19, 967.
Prosperi, E., 1997. Prog. Cell Cycle Res. 3, 19.
Gulbis, J., et al. 1996. Cell, 87, 297.
Brott, D.A., et al. 1993. J. Cell. Biochem. 52, 362.
User Protocol

Revision07-March-2011 JSW
Form96 Tests
Format96-well strip plate
Detection methodColorimetric
Specieshuman, mouse, rat
StorageUpon receipt of the kit, standards must be stored at -20°C. All other components may be stored at 4°C. Do not expose reagents to excessive light. Let kit sit at room temperature for 30 min before use. Note: This kit contains an overnight step during sample preparation.
Intended useThe Calbiochem® brand PCNA ELISA is a non-isotopic immunoassay for the in vitro quantitation of human, rat and mouse PCNA protein in cell lysates, tissue culture medium, sera and plasma.
BackgroundProliferating Cell Nuclear Antigen (PCNA) has no known enzymatic activity; it can, however, interact with a variety of proteins and regulate their activities. PCNA and its adapter protein replication factor C (RFC); cooperate to form a moving clamp that is an attachment point for DNA polymerases delta and epsilon. PCNA is central to a decision pathway between a diverse array of important cellular processes including cell cycle control, DNA replication, nucleotide excision repair, post-replication mismatch repair, and at least one apoptotic pathway. The perpetual transfer of proliferating cell nuclear antigen on and off the DNA renders this protein an ideal site for a variety of protein interactions that are essential for DNA metabolic events. PCNA is an essential component of the DNA-replication and repair machinery as shown by its interactions with enzymes that are important for nucleic-acid metabolism, including DNA replication, DNA repair and post-replication processes. Besides forming a homotrimeric structure which circles the DNA and loads the polymerase on the DNA template, PCNA is also capable of binding to other proteins, including the FEN1/Rad27/MF1 nuclease, DNA ligase 1, the nucleotide excision repair protein XPG, DNA-cytosine-5 methyltransferase, and the mismatch repair proteins MLH1 and MSH2. A role for PCNA in cell cycle control is recognized by its interaction with cell-cycle regulators that play multiple roles in cell-cycle progression and checkpoint control proteins such as cyclins, cyclin-dependent kinases (cdks) and the CDK-inhibitor p21waf1/cip1/sdi1 protein. In addition, the role of PCNA as a component of the cell cycle control apparatus is demonstrated by its association with the growth-arrest and DNA-damage inducible proteins gadd45 (p53-inducible) and MyD118. These varied interactions with DNA metabolic proteins imply that PCNA is a central factor for the coordination of DNA replication, DNA repair, epigenetic inheritance, and cell-cycle control. The trimeric ring-like structure of PCNA is loaded onto DNA at a primer-template junction or onto a nicked site in duplex DNA by RFC. PCNA functions as a processivity factor for polymerase delta during DNA replication. PCNA loading is a prerequisite for assembly of polymerase delta onto the template DNA to form a processive holoenzyme, which then functions during synthesis of both the leading and lagging strands at a DNA replication fork. PCNA is also believed to be involved in the function of DNA polymerase epsilon. Since PCNA is present at very low levels in quiescent cells it can be a significant marker for detecting the transition of a non-proliferating cell population to a proliferating cell population. Even though PCNA has been shown to be stable during the cell cycle, in transformed cell lines it is still possible to show that PCNA levels are lower in non-proliferating cells provided that the cells remain quiescent for several doubling times. The primary amino acid sequence of PCNA is not highly conserved among species, but yeast and human PCNA nevertheless have an almost identical three-dimensional shape. E. coli (DNA polymerase III) and bacteriophage T4 (gp45 protein) are functional homologues of PCNA and share the same three-dimensional closed ring structure with a hole in the center that encircles duplex DNA.
Principles of the assayThe PCNA ELISA is a "sandwich" enzyme immunoassay employing the mouse monoclonal antibody clone PC10 and a rabbit polyclonal antibody. A polyclonal antibody, specific for the human PCNA protein, has been immobilized onto the surface of wells provided in the kit. The sample to be assayed and the biotinylated detector monoclonal antibody are pipetted into the wells and allowed to incubate for 2 h, during which time any PCNA present binds to the capture and detecting antibodies. Unbound material is washed away and horseradish peroxidase-conjugated streptavidin is added, which binds to the detector antibody.

The horseradish peroxidase catalyzes the conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stop reagent), the intensity of which is proportional to the amount of PCNA protein in the sample. The colored reaction product is quantified using a spectrophotometer.

Quantitation is achieved by the construction of a standard curve using known concentrations of PCNA (provided lyophilized). By comparing the absorbance obtained from a sample containing an unknown amount of PCNA with that obtained from the standards, the concentration of PCNA in the sample can be determined.

The PCNA ELISA has the following characteristics:
• Correlates with BrdU Cell Proliferation Assay, Cat. No. QIA58.
• Can detect PCNA in human, mouse and rat cells.
• Has excellent sensitivity and precision.
• Has a short and simple format.
• Can detect PCNA in cell lysates.
• Measures PCNA in cell culture supernatants.
• Detects PCNA in auto-immune sera samples.
Materials providedStandards should be assayed in duplicate. A standard curve must be performed on the same plate and at the same time as the samples. The PCNA ELISA provides sufficient reagents to run two sets of standard curves, and 41 samples (if assayed in duplicate all at once using one standard curve), or 34 samples (if assayed on two separate occasions using two standard curves).

• Anti-PCNA coated 96 well plate (Kit Component No. JA1848-1EA): 96 removable wells coated with PCNA polyclonal antibody.
• PCNA Standard (Kit Component No. JA1849-1EA): two vials containing lyophilized PCNA protein (please refer to the detailed protocol below and the vial label for reconstitution instructions).
• Reconstituted standards should be discarded after one use.
• Detector Antibody (Kit Component No. JA1851-6ML): biotinylated monoclonal anti-human PCNA antibody, clone PC10.
• 400X Conjugate (Kit Component No. JA1850-.1ML): Streptavidin-Peroxidase Conjugate; 400-fold concentrated solution.
• Conjugate Diluent (Kit Component No. JA1615-12ML): buffer for dilution of 400X Conjugate.
• Substrate (Kit Component No. JA1608-12ML): chromogenic substrate (TMB).
• Sample Diluent (Kit Component No. JA1852-25ML): buffer used to dilute standards and samples.
• 20X Plate Wash Concentrate (Kit Component No. JA1617-100ML): 20-fold concentrated solution of PBS and surfactant. Contains 2% chloroacetamide.
• Antigen Extraction Agent (AEA) (Kit Component No. JA1855-3ML): two vials; use as directed in sample preparation section for the extraction of PCNA from cell preparations.
• Stop Solution (Kit Component No. JA1616-12ML): 2.5 N sulfuric acid.
• Plate Sealers: to cover plates during incubations.
Materials Required but not provided• 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips.
• Automated plate washer, wash bottle or multichannel dispenser for washing.
• 1 liter graduated cylinder.
• Deionized or distilled H2O.
• 0.2 µm syringe filter and syringe.
• Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/595 nm or 450/540 nm. A single wavelength of 450 nm can also be used.
• Cell Resuspension Buffer: 50 mM Tris, containing 5 mM EDTA, 0.2 mM PMSF, 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin adjusted to pH 8.0.
Precautions and recommendations• Store standards at -20°C; store all other components at 4°C. Do not expose reagents to excessive light.
• Let the kit sit at room temperature for 30 min before use. Best results will be obtained using reagents at room temperature.
• Wear disposable gloves and eye protection.
• Always use clean well-rinsed glassware. Soap residue may compromise assay performance.
• Use only the wells provided with the kit.
• Do not mix reagents from different kits.
• Do not mouth pipette or ingest any of the reagents.
• The buffers and reagents used in this kit contain anti-microbial and anti-fungal reagents. Care should be taken to prevent direct contact with these products.
• Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled.
• Human samples may be contaminated with infectious agents. Do not ingest, expose to open wounds, or breathe aerosols. Dispose of samples properly.
PreparationCell Lysate Protocol. Numerous extraction protocols can be used. The following protocol has been shown to work with a number of cell lines. It is provided as an example of a suitable extraction procedure, but should not be construed as necessarily being the method of choice. Users may wish to experiment with extraction procedures that work best in their laboratory. 1. For suspension cells, pellet by centrifugation, remove supernatant, resuspend with PBS and pellet by centrifugation. For attached cells, remove supernatant from cells (you may save the supernatant for testing in the ELISA). Wash cells once with PBS, harvest cells by scraping and gentle centrifugation. 2. Aspirate PBS leaving an intact cell pellet (at this point the cell pellet can be frozen at -80°C and lysed at a later date). We recommend for every 5 x 106 cells, resuspend the pellet in 1 ml of Resuspension Buffer. 3. Add 20 µl of Antigen Extraction Agent (AEA provided) for every 100 µl of cell suspension. 4. Incubate 30 min on ice with occasional vortexing. 5. Transfer extracts to microcentrifuge tubes and centrifuge for 5 min at 500 x g at 4°C. 6. Aliquot cleared lysate to clean microfuge tubes. The sample should be aliquotted to avoid multiple freeze/thaws. Samples must be frozen at -20°C for 18-24 h prior to testing for PCNA levels in the assay. These samples are now ready for analysis according to the instructions provided in the Detailed Protocol. Samples may be stored at -20°C for future testing in the PCNA ELISA. Samples found to contain greater than 30 units/ml PCNA (i.e., outside the range of the standard curve) must be diluted with Sample Diluent (provided), so that the PCNA concentration falls within the range spanned by the standard curve, and assayed again. Format for Biological Experiments Designed using 96-Well Tissue Culture Plates. Grow cells in desired media and plate cells at desired concentration (100 µl/well) in a sterile 96-well tissue culture plate. The PCNA ELISA is sensitive enough to detect PCNA levels from 0.625 x 104 cells/well. Treat cells with drug(s) of choice at the appropriate dose and time courses. • Prepare Resuspension Buffer (50 mM Tris, containing 5 mM EDTA, 0.2 mM PMSF, 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin adjusted to pH 8.0). You will need ~40 µl per cell well, so multiply the number of wells you have by 40 to obtain the volume of resuspension buffer needed. • Add 1 part Antigen Extraction Agent (AEA) supplied in the kit to 5 parts resuspension buffer. This is your Lysis Buffer. • To harvest the cells, spin the 96-well plate at 500 x g for five min. Remove the supernatant by emptying contents into a reservoir (or save supernatant if you wish to test for PCNA levels). Blot the plate dry. • Immediately lyse the cells with 40 µl/well Lysis Buffer. Pipette the liquid in each well several times to ensure the best extraction possible. Incubate the plate(s) on ice for 30 min. • Gently shake plate and wrap in parafilm. Place the plate at -20°C for at least 2 h, or until the cell extracts in the wells are completely frozen. The plates may be stored at -20°C for future testing in the PCNA ELISA. • Thaw the 96-well plate extracts at room temperature. • Add 160 µl of Sample Diluent per well and mix gently (performing a 1:5 dilution of your samples right in the cell wells). Your samples are now ready to be run in the ELISA. It is convenient to simply transfer the samples directly to the ELISA (50 µl/well) since they are already diluted 1:5 in the cell wells. However, you may dilute them further in sample diluent as needed. • Culture Medium Protocol. For suspension cells: Pellet by centrifugation (1000 x g for 10 min, 4°C) and remove supernatant for testing. Samples may be stored at -20°C. • For adherent cells: Remove tissue culture media, centrifuge tissue culture media (10,000 x g for 10 min), and remove supernatant for testing. Samples may be store at -20°C.
Detailed protocolThe PCNA ELISA is provided with removable strips of wells so the assay can be carried out on two separate occasions. Since conditions may vary, a standard curve must be determined each time the assay is performed. Standards should be assayed in duplicate. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross-contamination of reagents or samples.
1. Remove the appropriate number of wells from the foil pouch. Return any unused wells to the foil pouch, reseal and store at 4°C. Let all other kit components sit at room temperature until used. Best results will be obtained using reagents at room temperature.
2. Prepare a working solution (1X) of Wash Buffer by adding 50 ml of the 20X concentrated solution (provided), to 950 ml of deionized water. Mix well.
3. Each time an assay is performed, reconstitute a Lyophilized Standard by carefully and accurately pipetting dH2O and/or sample diluent, as described on the lyophilized PCNA Standard vial label to give a concentration of 200 ng/ml. Let the reconstituted standard sit for 15 min at room temperature, with occasional swirling. Avoid excessive agitation of the standard. After reconstituting the PCNA Standard it should be diluted with Sample Diluent. Obtain eight tubes and label them 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0 ng/ml. Add 250 µl of Sample Diluent into each tube except the 200 ng/ml tube (first tube) which gets "undiluted" reconstituted standard. Remove 500 µl from the original vial of lyophilized material and add it to the first tube. Remove 250 µl from the first tube (200 ng/ml) and add it to the second tube (100 ng/ml) and mix gently. Repeat this procedure until you reach the seventh tube (3.125 ng/ml). The last tube (0 ng/ml) should just be Sample Diluent. Reconstituted standards should be discarded after one use.
4. Prepare all samples. A recommended starting dilution for all samples is a 1:5 dilution with sample diluent.
5. Pipette 50 µl of the Detector Antibody into each well.
6. Add samples and each of the PCNA standards (in duplicate) by pipetting 50 µl into appropriate wells using clean pipette tips for each sample.
7. Cover wells with a plate sealer and incubate at room temperature for 2 h.
8. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely.
9. Dilute a sufficient amount of the 400X Conjugate 1:400 in Conjugate Diluent to provide 100 µl of 1X solution for each sample and standard well (For example: add 30 µl to 11.970 ml of Conjugate Diluent), mix gently. Filter with a 0.2 µm syringe filter prior to use. Filtering will reduce background.
10. Pipette 100 µl of the 1X Conjugate into each well, cover with a plate sealer and incubate at room temperature for 30 min. Discard any unused 1X Conjugate.
11. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely.
12. Flood entire plate with dH2O. Remove contents of wells by inverting over sink and tapping on paper towels.
13. Add 100 µl of Substrate Solution to each well and incubate in the dark at room temperature for 30 minutes.
14. Add 100 µl of Stop Solution to each well in the same order as the previously added Substrate Solution.
15. Measure absorbance in each well using a spectrophotometric plate reader. It is preferable to read at dual wavelengths of 450/595 nm (or 450/540 nm). A single wavelength of 450 nm can also be used. Wells must be read within 30 min of adding the Stop Solution.
Calculations1. Average the duplicate absorbance values for each standard, including the zero, and all sample values. 2. On graph paper, plot the mean absorbance values for each of the standards on the Y axis, versus the concentration of each standard (ng/ml) on the X axis. 3. Determine the concentration of unknowns by interpolation from the standard curve. There are a variety of plate reader software packages available (Softmax, Molecular Devices Corporation, Menlo Park, CA; KinetiCalc, BioTek Instruments, Inc. Winooski, VT) for analysis of plate data, which simplifies this process. 4. For samples which have been diluted, the PCNA concentration must be multiplied by the dilution factor (ie., if the sample was diluted five-fold, then the PCNA value obtained from the standard curve must be multiplied by five).
Standard curve

Figure 1: Standard Curve

The mean signal of each standard run in replicates of two in four assays using two different batches of plates and two different lots of detector antibody. Note: 44 units/ml of the old standard (lysate) corresponds to about 200 ng/ml of the new standard.
Example data

Figure 2: Proliferating Cell Nuclear Antigen

Proliferating Cell Nuclear Antigen (PCNA) Levels and BrdU Incorporation in Human Peripheral Blood Lymphocytes (PBLs) After Stimulation With Phytohemagglutin (PHA). PBLs were induced to proliferate with 25 µg/ml PHA for 72 h. Aliquots of stimulated and unstimulated cells were diluted to 2 x 105 cells/ml and plated at 100 µl/well in a microtiter plate. The samples were incubated with BrdU for 2 h at 37°, 7% CO2. Identical aliquots were assayed for PCNA with the PCNA ELISA (Cat. NO. QIA59).

Figure 3: PCNA ELISA

Detection Of Changes In PCNA Levels In Human Peripheral Blood Lympho-cytes After Stimulation with PHA by the PCNA ELISA (Cat. No. QIA59).

Figure 4: Detection of Changes in PCNA Levels

Detection Of Changes In PCNA Levels After Phorbol Ester (PMA) Induced Growth Arrest by the PCNA ELISA (Cat. No. QIA59). Macrophage-like differentiation can be induced in HL-60 cells by PMA. HL-60 cells were plated at 2 x 104 cells per well and various concentrations of PMA were added for a dose-response study. Cells were incubated for 96 h, then assayed as described.

Figure 5: Concanavalin A Induced Up-Regulation of PCNA Protein

Concanavalin A Induced Up-Regulation Of PCNA Protein In Mouse Splenocytes. Mouse splenocytes were incubated at 2 x 106 cells/ml for 72 h with various concentrations of Concanavalin A (ConA). Samples were then assayed as described.

Figure 6: Concanavalin A Induced Up-Regulation of PCNA Protein

Concanavalin A induced up-regulation of PCNA protein in Rat Splenocytes. Rat splenocytes were incubated at 2 x 106 cells/ml for 48 h with various concentrations of Concanavalin A. Samples were prepared and tested in the PCNA ELISA.

Figure 7: Phytohemagglutin induced up-regulation of PCNA protein

Phytohemagglutin induced up-regulation of PCNA protein in Rat Splenocytes. Rat splenocytes were incubated at 2 x 106 cells/ml for 48 h with various concentrations of PHA. Samples were prepared using the Cell Lysate Protocol and tested in the PCNA ELISA.
Assay Range3.125-200 ng/ml of PCNA
Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
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