p21 Human ELISA kit For the quantitative determination of Human p21 in cell lysates.

Transcription

1 ab p21 Human ELISA kit Instructions for Use For the quantitative determination of Human p21 in cell lysates. This product is for research use only and is not intended for diagnostic use.

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3 Table of Contents 1. Introduction 3 2. Principle of the Assay 4 3. Assay Summary 5 4. Kit Contents 6 5. Storage and Handling 7 6. Additional Materials Required 7 7. Protocol 8 8. Calculation of Results Performance Characteristics Troubleshooting 22 2

4 1. Introduction ab p21 Human ELISA kit is a complete kit for the quantitative determination of Human p21 in cell lysates. Please read the complete kit insert before performing this assay. Progression through the cell cycle is controlled by cyclin-dependent kinases (CDKs). Human p21waf1/cip1 is a CDK inhibitor that was first cloned and characterized as a mediator of p53-induced growth arrest. Human p21 is a 164 amino acid nuclear protein with a calculated molecular weight of 18,119 Daltons whose expression is p53-dependent. Human p21 is found in a complex involving cyclins, CDKs and PCNA in normal cells but not transformed cells. Human p21 may also be upregulated independently of p53 by TGF-β, tamoxifin, progesterone, nerve growth factor and apicidin. Human p21 regulates cell cycle progression, terminal differentiation and apoptosis. Human p21 expression has been implicated in a variety of Human cancers including those of the prostate, bladder and esophagus. Increased expression of the HOXB4 transcription factor in combination with suppression of p21 expression could be a useful strategy for effective and robust expansion of hematopoetic stem cells. 3

5 2. Principle of the Assay 1. Standards and samples are added to wells coated with a monoclonal antibody specific for Human p21. The plate is then incubated. 2. The plate is washed; leaving only bound Human p21 on the plate. A yellow solution of polyclonal antibody to Human p21 is then added. This binds the Human p21 captured on the plate. The plate is then incubated. 3. The plate is washed to remove excess p21 antibody. A blue solution of HRP conjugate is added to each well, binding to the human p21 polyclonal antibody. The plate is again incubated. 4. The plate is washed to remove excess HRP conjugate. TMB Substrate solution is added. The substrate generates a blue color when catalyzed by the HRP. 5. Stop solution is added to stop the substrate reaction. The resulting yellow color is read at 450 nm. The amount of signal is directly proportional to the level of Human p21 in the sample. 4

6 3. Assay Summary Sample and standards are added to the pre-coated well coated with Mouse monoclonal antibody specific to Human p21. Incubate Wash Human p21 EIA Antibody Incubate Wash Human p21 EIA Conjugate Incubate Wash Add TMB Substrate Incubate Add Stop Solution Read Plate (measure at 450 nm) 5

7 4. Kit Contents Item Description Quantity Human p21 Clear Microtiter Plate Assay Buffer RIPA Cell Lysis Buffer 2 Human p21 Standard Human p21 ELISA Conjugate Human p21 ELISA Antibody Wash Buffer Concentrate (10X) A clear plate of break apart strips coated with Mouse monoclonal antibody specific to Human p21. Tris buffered saline containing detergents. Tris-HCL containing detergents One vial containing 40,000 pg/ml of recombinant Human p21. A blue solution of Goat anti-rabbit IgG conjugated to horseradish peroxidase. A yellow solution of polyclonal antibody to Human p21. Tris buffered saline containing detergents. TMB Substrate A solution of 3,3 5,5 tetramethylbenzidine (TMB) and hydrogen peroxide. Stop Solution 2 Plate Sealer A 1N solution of hydrochloric acid in water. 1x 96 wells 30 ml 100 ml 1 vial 10 ml 10 ml 100 ml 10 ml 10 ml 3 units 6

8 5. Storage and Handling All components of this kit, except the Standard, should be stored at 4 C upon receipt. The Standard should be stored at 20 C. 6. Additional Materials Required Deionized or distilled water. Precision pipets for volumes between 5 μl and 1000 μl. Repeater pipets for dispensing 100 μl. Disposable beaker for diluting buffer concentrates. Graduated cylinders. A microplate shaker. Lint free paper toweling for blotting. Microplate reader capable of reading at 450 nm. Graph paper for plotting the standard curve. Disposable polypropylene tubes for dilution of samples and standards. Phenylmethylsulfonylfluoride (PMSF). Protease inhibitor cocktail (PIC) - not containing AEBSF. 7

9 7. Protocol A. Sample Handling The assay is compatible with Human p21 samples in a wide range of cell lyates. Prior to assay, frozen samples should be brought slowly to 4 C (on ice) and centrifuged, if necessary, to isolate residual cell debris. Human p21 is susceptible to proteases and it is crucial to prevent contamination of the reagents. A minimum 1/40 dilution is required for RIPA Cell Lysis Buffer 2 to remove matrix interference of this buffer. Due to differences in cell types, number of cells, or total cellular protein concentration, lysates may require a greater dilution with assay buffer plus inhibitors to remove interference or to be read within the standard range. B. Reagent Preparation 1. Wash Buffer Prepare the wash buffer by diluting 50 ml of the supplied Wash Buffer Concentrate with 950 ml of deionized water. This can be stored at room temperature for 3 months. 8

10 2. Human p21 Standards Allow the 40,000 pg/ml Human p21 standard solution to warm to room temperature. Label seven 12 x 75mm tubes #1 through #7. Pipet 975 μl of Assay Buffer plus inhibitors into tube #1. Pipet 500 μl of Assay Buffer plus inhibitors into tubes #2 through #7. Add 25 μl of the 40,000 pg/ml standard into tube #1 and vortex thoroughly. Add 500 μl of tube #1 to tube #2 and vortex thoroughly. Add 500 μl of tube #2 to tube #3 and vortex thoroughly. Continue this for tubes #4 through #7. Diluted standards should be used within 30 minutes of preparation. The concentration of Human p21 in tubes #1 through #7 will be 1000, 500, 250, 125, 62.5, 31.25, and pg/ml respectively. 3. Assay Buffer Prepare the Assay Buffer by diluting 25 ml of the supplied concentrate with 100 ml of deionized water. This can be stored at room temperature for 3 months. 4. PIC and PMSF Addition Immediately prior to use, PIC and PMSF must be added to the assay buffer and RIPA Cell Lysis Buffer 2. Add 0.5 μl/ml or the equivalent concentration of AEBSF-free PIC 9

11 according to the vendor s specification sheet. Add PMSF, to a final concentration of 1mM. Each inhibitor treated buffer should incubate for 5-10 minutes at room temperature prior to use. Buffers treated with inhibitors should be used within 1 hour of preparation. C. Assay Procedure Refer to the Assay Layout Sheet to determine the number of wells to be used. Remove the wells not needed for the assay and return them, with the desiccant, to the mylar bag and seal. Store unused wells at 4 C. 1. Pipet 100 μl of the standard diluent into the S0 (0 pg/ml standard) wells. 2. Pipet 100 μl of the samples to the bottom of the appropriate wells. 3. Pipet 100 μl of Standards #1 through #7 to the bottom of the appropriate wells. 4. Seal the plate. Incubate for 1 hour on a plate shaker (~500 rpm*) at room temperature. 10

12 5. Empty the contents of the wells and wash by adding 400 μl of wash buffer to every well. Repeat 4 more times for a total of 5 washes. After the final wash, empty or aspirate the wells and firmly tap the plate on a lint free paper towel to remove any remaining wash buffer. 6. Pipet 100 μl Human p21 ELISA Antibody into each well except the blank. 7. Seal the plate. Incubate for 1 hour on a plate shaker (~500 rpm*) at room temperature. 8. Wash as above (Step 5). 9. Add 100 µl of Human p21 ELISA Conjugate to each well except the blank. 10. Seal the plate. Incubate for 1 hour on a plate shaker (~500 rpm*) at room temperature. 11. Wash as above (step 5). 12. Add 100 μl of the TMB substrate solution into each well. 13. Incubate for 30 minutes at room temperature on a plate shaker (~500 rpm*) at room temperature. 14. Pipet 100 μl of stop solution into each well. 11

13 15. After blanking the plate reader against the substrate blank, read optical density at 450 nm. If plate reader is not capable of adjusting for the blank, manually subtract the mean OD of the substrate blank from all readings. *The optimal speed for each shaker will vary and may range from rpm. The speed must be set to ensure adequate mixing of the wells, but not so vigorously that the contents of the wells splash out and contaminate other wells. 12

14 Assay Layout Sheet: A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 Blank Std 3 Std 7 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 Blank Std 3 Std 7 C1 S0 C2 Std 4 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 D1 S0 D2 Std 4 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 E1 Std 1 E2 Std 5 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 F1 Std 1 F2 Std 5 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 G1 Std 2 G2 Std 6 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 H1 Std 2 H2 Std 6 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 13

15 8. Calculation of Results Several options are available for the calculation of the concentration of Human p21 in the samples. We recommend that the data be handled by an immunoassay software package utilizing a 4 parameter logistic (4PL) curve fitting program. If data reduction software is not readily available, the concentrations can be calculated as follows: 1. Calculate the average Net OD for each standard and sample by subtracting the average blank OD from the average OD for each standard and sample. Average Net OD = Average OD - Average Blank OD 2. Using linear graph paper, plot the average Net OD for each standard versus Human p21 concentration in each standard. Approximate a straight line through the points. The concentration of the unknowns can be determined by interpolation. Samples with concentrations outside of the standard curve range will need to be re analyzed using a different dilution. 14

16 Typical Results The results shown below are for illustration only and should not be used to calculate results. Sample Net OD Human p21 (pg/ml) S S ,000 S S S S S S Unknown Unknown

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18 9. Performance Characteristics A. Specificity The cross reactivities for a number of related compounds were determined by diluting the potential cross reactants in Assay Buffer 23 at 100,000 pg/ml. These samples were then measured in the assay. Compound Cross Reactivity ciap1 <0.02% p38 <0.02% pmek <0.02% JNK <0.02% β - Catenin <0.02% p300 <0.02% Smac / DIABLO <0.02% 17

19 B. Sensitivity The sensitivity, defined as the concentration of Human p21, measured at 2 standard deviations from the mean of 24 zeros along the standard curve, was determined to be 8.0 pg/ml. C. Linearity 1. A lysate from apicidin treated HeLa cells was diluted 1/40 and then further diluted serially. The results are shown in the table below. Dilution Expected (pg/ml) Observed (pg/ml) Recovery (%) 1/ / / / /

20 2. A buffer sample containing Human p21 was serially diluted 1/2 in kit Assay Buffer 23 and measured in the assay. The results are shown in the table below. Dilution Expected (pg/ml) Observed (pg/ml) Recovery (%) 1/ / / / / D. Precision 1. Intra assay precision was determined by assaying 20 replicates of three buffer controls containing Human p21 in a single assay. pg/ml %CV

21 2. Inter assay precision was determined by measuring buffer controls of varying Human p21 concentrations in multiple assays over several days. pg/ml %CV D. Stimulation Response HeLa cells were stimulated with 2 μg/ml apicidin or DMSO for 24 hours. Cells were harvested, washed and cell pellets were resuspended with RIPA Cell Lysis Buffer 2 plus inhibitors at 4 x 10 6 cells/ml (~1 mg/ml total cellular protein). The cells were vortexed and lysed on ice for 30 minutes. The lysates were vortexed and pelleted at 16,000 x g for 20 minutes at 4 C. The supernatants were either assayed immediately by Western analysis and ELISA or stored at - 70 C. The results of the Western blotting and ELISA are shown below. Apicidin treatment resulted in a 4.4 fold increase in p21 levels by ELISA that mirrors the increase observed by Western blotting. 20

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23 10. Troubleshooting Weak Color Development How long was the substrate incubation? What were the conditions of the substrate incubation? Were reagents brought to room temperature prior to use? It is possible that Stop Solution was added to the plate without allowing the full substrate incubation. If a plate is left to incubate on a cold lab bench or under a drafty area during ambient incubations, signal values (e.g. optical density) may be lower than expected. It is important to ensure that all reagents are brought to room temperature prior to use, or as mentioned in the product specific instruction manual. Usually leaving the kit out on the bench top at ambient temperature for about half an hour prior to setting up the assay will be sufficient, when the reagents can be stored at 4 C. Frozen volumes take a little more time to come to room temperature. Do not thaw frozen reagents in a water bath. If a different standard/sample diluent is used (such as culture media) this must also be warmed. 22

24 What were the conditions of the incubations? How was the plate shaken during incubations (if required)? How long after the addition of Stop Solution was the plate read? If the incubation times and temperatures are not observed, this can lead to lower than expected signal values (e.g. optical density). Pay attention that in airconditioned rooms the temperature does not drop below 21 C. If customers do not have a plate shaker, they will often use an orbital flask shaker or some other piece of equipment. This is not a problem as long as the liquid is vigorously displaced about 3/4 of the way up the sides of the wells without coming out. It is very important that the plate is secured into place. If the plate is not shaken and it is required in the procedure, a longer incubation may be necessary to bring the reagents to equilibrium. The plate needs to be read at the correct wavelength as soon as possible after the addition of the Stop Solution. We generally recommend that the plate be read within 10 minutes. 23

25 High Background How was the plate washed? What were the incubation times and temperatures? It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The plate contents can be dumped into the sink and shaken to remove excess buffer. This should be repeated for the number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels. If the plate was incubated for too long or at a higher than recommended temperature, high background could result. 24

26 Drift Were reagents brought to room temperature prior to use? Was the set-up of the assay interrupted? If the reagents are not at a constant temperature prior to their addition into the wells, the results from one side of the plate to the other can differ depending on the temperatures at addition. If the assay is interrupted at any point during the addition of reagents, it is possible that differing results will be seen before the interruption versus after. The wells that had reagents added before the interruption will have been incubating for longer than those after. 25

27 Poor Precision Were the wells washed properly? All wells receive the same treatment during the wash step. If some are washed less than others, this can translate to poor precision. It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The plate contents can be dumped into the sink and shaken to remove excess buffer. This should be repeated for the number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels. 26

28 Were the wells aspirated sufficiently after the wash steps? How were reagents pipetted into wells? It is very important that as little Wash Buffer as possible remains in the wells after aspiration. Residual buffer can cause dilution of subsequent reagents. After the last wash step, it is a good idea to hit the plate several times over a piece of paper toweling to remove excess buffer. In order to eliminate precision error, customers need to remember to prerinse all pipet tips used in the assay. We usually recommend that the customer draw up the liquid into the tip and aspirate it three times prior to addition into the well. Regular pipet calibration and maintenance is also essential to ensure that the tips fit properly and that the correct volumes are dispensed. Be sure reagents are not splashed between wells or outside of the wells during pipeting (especially if using repeater pipets). 27

29 Poor Standard Curve What was used as the standard diluent? How was the precision of the standard curve? Were the Blank and NSB values subtracted out? Diluents other than the supplied assay buffer may contain interfering substances that can affect the standard curve. If the %CV values for the standard curve signal values (e.g. optical density) are consistently above 5%, it may be a good idea to pay particular attention to pipetting technique. If the standard curve signal values were acceptable but the sample precision was not, the problem relates to the sample. Also, see recommendations under "Poor Precision". If the net signal values (e.g. optical density) are not used, the signal values will appear higher than those presented in the sample data in the instruction manual. 28

30 How were the standard dilutions prepared? It is important that test tubes of an appropriate size and material are used. Standard dilutions must be properly mixed (e.g. vortexed) while preparing the serial dilutions. It is also crucial that the standard dilutions be prepared and used within the time specified in the product specific instruction manual. Never store unused standard dilutions for a later use. 29

31 Edge Effects Where was the plate incubated? If multiple plates were run, were they stacked on top of each other during incubation? If a non-ambient incubation was required, was the plate properly sealed? Often times the conditions for ambient incubations can be less than ideal. If there is a draft in the area or the plate is incubated on a cold lab bench, this can lead to uneven color development. Multiple plates should only be incubated in a single layer. This will assure that no area of the plate is at a different temperature than any other. Making sure that the plate sealer is tightly covering all of the wells will help to discourage uneven evaporation of the well contents, or condensation for colder incubation conditions. 30

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