BIOO RESEARCH PRODUCTS. In Cell ELISA Kit Manual Catalog #:

Transcription

1 BIOO RESEARCH PRODUCTS In Cell ELISA Kit Manual Catalog #: BIOO Scientific Corp. 2008

2 TABLE OF CONTENTS GENERAL INFORMATION... 1 Product Description... 1 Procedure Overview... 1 Kit Contents, Storage and Shelf Life... 2 Required Materials Not Provided With the Kit... 2 Warnings and Precautions... 2 IN CELL ELISA KIT PROTOCOL... 3 Reagent Preparation... 3 In Cell ELISA Protocol... 4 TROUBLESHOOTING... 5 No color development or No Signals with Standards... 5 Low Optical Density (OD) Readings... 5 High Background or High Optical Density (OD) Readings... 6 High Intra-Plate or Inter-Plate Variance... 6 MaxDiscovery In Cell ELISA Kit is intended for laboratory use only, unless otherwise indicated. This product is NOT for clinical diagnostic use. MaxDiscovery is a Trademark of Bioo Scientific Corporation (BIOO).

3 GENERAL INFORMATION GENERAL INFORMATION Product Description The MaxDiscovery In Cell ELISA Kit is an enzyme immunoassay for quantitative protein analysis directly in adherent cell cultures there is no need to lyse cells or run protein gels. The technique can be thought of as an ELISA performed on fixed, permeablized cells rather than on immobilized antigen. Like most ELISA assays, protein is detected using a series of two antibodies, a primary antibody for the protein of interest (not provided with the kit) and an enzyme-conjugated secondary antibody. The protein:antibody complex is visualized by adding an enzyme substrate that, when catalyzed, changes the color of the assay solution within the well of the tissue culture plate so that it can be read in a spectrophotometer at 450 nm. The relative amount of protein in the cells will be directly proportional to the amount of signal that is obtained, making this a simple, convenient, and accurate technique for quantifying specific protein levels within cell lines. The MaxDiscovery In Cell ELISA Kit contains materials for washing, fixing, and permeablizing cells in culture. HRP-conjugated anti-mouse and anti-rabbit secondary antibodies are included in the kit so that user-supplied primary antibodies from either mouse or rabbit may be used for detection of the protein of interest. In addition, the kit includes mouse-derived antibody targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH is one of the key enzymes involved in glycolysis, and because of its high-level, and constitutive expression, it is widely used as a loading control for Northern/Western blots and for protein normalization. The quantity of GAPDH in a sample is directly proportional to the number of cells in the sample. GAPDH expression is often used as an internal control in gene expression studies. Typically, GAPDH expression is used to normalize expression signal from the gene-of-interest with respect to cell number. This has been particularly useful for RNAi studies or other drug treatment studies. The MaxDiscovery In Cell ELISA assay makes it easy to quickly measure levels of GAPDH (positive control) or your protein-of-interest directly in cultured cells for relative quantitative analysis. The unique features of the kit include: Rapid minute sample preparation measure protein directly in cells. Quantitative measurement of protein levels in cultured cells. Includes anti-gapdh antibody for signal normalization. Scalable (can be used for 6-well to 384-well plates, and all of the sizes in between). Get results in as few as 3 hours instead of the hours required for Western analysis. Ideal for cellular screening assays rapidly quantitate protein levels after treating cultured cells with drugs or small RNA molecules such as mirna or sirna. Procedure Overview The MaxDiscovery In Cell ELISA procedure requires living adherent cultured mammalian cells grown to ~80% confluency in multiwell culture plates. Cells may be manipulated in any fashion prior to analysis with the MaxDiscovery In Cell ELISA assay. In the first three steps of the procedure, the cells are washed, fixed, and permeablized. Once the cells are permeablized, primary antibody for the protein of interest (not supplied with the kit) is added to the cell samples. In general, any antibody that can be used for immunofluoresence or immunohistochemistry will be compatible with the MaxDiscovery In Cell ELISA assay. Secondary antibody, HRP-conjugated anti-mouse IgG or anti-rabbit IgG is added next. It targets the primary antibody bound to target in the cells attached to the plate wells. Next, 3, 3, 5, 5 -Tetramethylbenzidine (TMB), the substrate for HRP, is added. When oxidized, TMB turns blue, with major absorbance peaks at 370 nm and 652 nm. Finally, Stop Buffer is added to stop the HRP reaction and to change the reacted substrate from blue to yellow. Assay results are read in a spectrophotometer set to 450 nm. The intensity of the resulting color is directly related to the concentration of the target protein in the cells. A set of separate wells in the plate can be assayed for GAPDH, and the data can be used to normalize for protein concentration differences due to experimental treatment, facilitating a direct comparison of target protein levels from one well to another in a comparative quantitative fashion. BIOO RESEARCH PRODUCTS 1

4 Kit Contents, Storage and Shelf Life MaxDiscovery In Cell ELISA Kit Manual The MaxDiscovery In Cell ELISA Kit includes enough reagents for 2 x 96 well plates. Store the kit at 4 C*. The shelf life is 12 months when the kit and its components are properly stored. Kit Contents Amount Storage Cell Fixation Buffer 20 ml 4ºC* Cell Permeabilization Buffer 20 ml 4ºC 10X PBS 2 x 15 ml 4ºC Mouse anti-gapdh Antibody (Antibody #1) 5 µl 4ºC 100X HRP-Conjugated Anti-Mouse IgG Antibody #2 250 µl 4ºC* 100X HRP-Conjugated Anti-Rabbit IgG Antibody #2 250 µl 4ºC* Antibody #1 Diluent 25 ml 4ºC Antibody #2 Diluent 25 ml 4ºC 20X Wash Solution 28 ml 4ºC Stop Buffer 25 ml 4ºC TMB Substrate 2 x 12 ml 4ºC * If you are not planning to use the kit for > 3 months, we recommend storing all supplied antibodies and the Cell Fixation Buffer in a 20 C frost-free freezer. Required Materials Not Provided With the Kit Microwell plate reader (450 nm) 10, 20, 100 and 1000 µl pipettors, and tips Multichannel pipettor: µl (Optional) Warnings and Precautions BIOO strongly recommends that you read the following warnings and precautions to ensure your full awareness of ELISA techniques and other details you should pay close attention to when running the assays. More information can also be found in the Troubleshooting section. Periodically, optimizations and revisions are made to the kit and manual. Therefore, it is important to follow the protocol included with the kit. If you need further assistance, you may contact your local distributor or BIOO at techsupport3@biooscientific.com. Do not use the kit past the expiration date. Try to maintain a laboratory temperature of 20º 25ºC (68º 77ºF). Avoid running assays under or near air vents, as this may cause excessive cooling, heating and/or evaporation. Also, do not run assays in direct sunlight, as this may cause excessive heat and evaporation. Cold bench tops should be avoided by placing several layers of paper towel or some other insulation material under the assay plates during incubation. Make sure you are using only distilled, deionized water since water quality is very important. When pipetting samples or reagents into an empty microtiter plate, place the pipette tips in the lower corner of the well, making contact with the plastic. Cell Fixation Solution contains formaldehyde, which is toxic. We recommend performing this incubation in a fume hood and wearing a lab coat, appropriate gloves, safety glasses, and other protective gear when using this solution. BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product. BIOO RESEARCH PRODUCTS 2

5 IN CELL ELISA KIT IN CELL PROTOCOL ELISA KIT PROTOCOL Reagent Preparation IMPORTANT: Place all reagents at room temperature 20 25ºC (68 77ºF) for 1 2 hours before use so that they are at room temperature before starting the assay. Mix all reagents by gently inverting or swirling prior to use. Prepare the following solutions just prior to starting the assay. Prepare only enough diluted antibodies for the experiment(s) to be completed that day. Keep diluted antibodies at room temperature for up to 2 hr, then store at 4ºC for up to 1 day. Do not put reagents back into the original stock tubes/bottles. 1. Preparation of 1X HRP-Conjugated Antibody #2 (Antibody #2) Mix 1 volume of 100X HRP-Conjugated anti-mouse IgG Antibody #2 or 100X HRP-Conjugated anti-rabbit IgG Antibody #2 (Antibody #2) with 99 volumes of Antibody #2 Diluent. Note: Use anti-mouse HRP-Conjugated Antibody #2 for detection of primary antibodies derived from mouse, such as monoclonal antibodies; prepare anti-rabbit HRP-Conjugated Antibody #2 for detection of primary antibodies derived from rabbit, such as polyclonal serum. 2. Preparation of user-supplied primary antibody (Antibody #1) Use the dilution factor recommended by the manufacturer of the antibody for immunofluorescence experiments. If this information is not available, you must determine the proper dilution factor empirically (typically 1:1000 to 1:5000 are good starting points). 3. Preparation of 1X Mouse anti-gapdh Antibody (Antibody #1) Optional Mix 1 volume of Mouse anti-gapdh Antibody (Antibody #1) with 999 volumes of Antibody #1 Diluent. 4. Preparation of 1X Wash Solution Mix 1 volume of the 20X Wash Solution with 19 volumes of distilled water. 5. Preparation of 1X PBS Mix 1 volume of 10X PBS with 9 volumes of distilled water BIOO RESEARCH PRODUCTS 3

6 In Cell ELISA Protocol NOTE: The procedure is written for a 96-well plate format with adherent cells. If the relative expression of the protein is low, it may be necessary to grow cells in 48-well plates (or larger) in order to detect the antigen. For non-adherent cells spinning the plates between each aspiration step is required. Cell Growth and Permeabilization 1. Seed cells in a 96-well plate so that they will be approximately 80% confluent at the time that the assay is started (for example, at a time to coincide with assessment of RNAi-mediated gene silencing or drug treatment). The growth area in each well of a 96-well plate is 0.32 cm Remove the tissue culture growth media. 3. Wash the cells by adding 100 µl 1X PBS to each well, swirl or tap the plate gently for about 20 seconds, then thoroughly remove all fluid by aspiration. 4. Fix cells by adding 100 µl Cell Fixation Buffer to each sample well. Incubate for 7 minutes at room temperature. Mix by gently swirling or tapping the plate. To minimize the escape of formaldehyde vapors from the Cell Fixation Solution, place the plate in a zip-lock bag. 5. Remove fixation solution and discard. 6. Wash each sample well 3 times with 250 µl 1X PBS, as described above. 7. Add 100 µl Cell Permeabilization Buffer to each sample well and mix by gently swirling or tapping the plate. Incubate at room temperature for 8 minutes. 8. Remove Cell Permeabilization Solution and discard. 9. Wash each well 3 times with 250 µl 1X PBS, as described above. Protein Detection 1. Add 100 µl diluted primary antibody (user supplied Antibody #1 or Anti-GAPDH Antibody #1) to each sample well, mix by gently swirling or tapping the plate and incubate at room temperature (20 25ºC / 68 77ºF) for 1 hour. 2. Remove primary antibody and wash the sample wells 3 times with 250 µl 1X Wash Buffer. After the last wash, invert the plate and gently tap the plate on paper towels to remove residual fluid. ( Proceed immediately to the next step. Do not allow the plate to air dry between steps.) 3. Add 100 µl secondary antibody (diluted in Antibody #2 Diluent), mix by gently swirling or tapping the plate and incubate at room temperature for 1 hour. 4. Wash the plate 3 times with 250 µl 1X Wash Solution. After the last wash, invert the plate and gently tap the plate on paper towels to remove residual fluid. ( Proceed immediately to the next step. Do not allow the plate to dry before adding the TMB substrate.) 5. Add 100 µl TMB substrate and cover the reaction plate. Begin timing the reaction immediately after adding the TMB substrate. Gently rock the plate manually for 1 minute to thoroughly mix the reaction. ( To avoid potential contamination, do not return TMB substrate to its original container once it has been removed. The TMB substrate should be a clear solution before it is added to an MaxDiscovery In Cell ELISA reaction; if the solution is colored, it indicates that the solution has deteriorated and should be discarded.) 6. Incubate for 5 45 minutes at room temperature, and then add 100 µl Stop Buffer to stop the enzyme reaction. The typical incubation time is 15 minutes but this depends on cell number, antibody affinity and the concentration of the target protein. 7. Read the plate on a plate reader set to 450 nm wavelength as soon as possible after adding the Stop Buffer ( Before reading, clean the bottom of the plate using a lint-free wipe to remove moisture or fingerprints that could interfere with accurate data collection.) BIOO RESEARCH PRODUCTS 4

7 % GAPDH protein expression Detection of sirna-mediated Knockdown of Gene Expression Using the MaxDiscovery In Cell ELISA Kit GAPDH sirna Negative control sirna Figure 1: Detection of Gene Knockdown Using the MaxDiscovery In Cell ELISA Kit. SKOV3 cells (6,000 cells/well), a cervical cancer cell line, were reverse transfected with an sirna targeting either GAPDH or a negative control sirna, using 0.6 µl of MaxSuppressor Cell Culture RNA-LANCEr in a 96-well plate. 72 hours after transfection, the MaxDiscovery In Cell ELISA Kit was used to detect GAPDH knockdown. TROUBLESHOOTING No color development or No Signals with Standards Possible Causes Recommended Action Reagents were used in the wrong order or a step was skipped. Wrong antibody was used, or antibody was prepared incorrectly or has deteriorated. TMB substrate has deteriorated or contaminated with HRP Follow the protocol carefully and repeat the assay. Make sure that the antibody used is the one that comes with the kit. The antibody is kit- and lot-specific. Make sure that the correct antibody and diluent are mixed in correct volumes. Use a new set of BIOO TMB substrate. Low Optical Density (OD) Readings Possible Causes Recommended Action Reagents were expired or mixed with a different lot number or were prepared incorrectly. Wash solution was prepared incorrectly. Too many wash cycles were used. Incubation times were too short. Lab temperature was too low. Reagents and plates were too cold. Reader was at wrong wavelength, or reader was malfunctioning. Excessive kit stress has occurred. Protein expression is low. Cell number is low. TROUBLESHOOTING Verify the expiration dates and lot numbers and that the reagents were prepared correctly. Use the wash solution for the kit and verify that it is prepared correctly. Make sure to use the number of washes per the protocol instruction. Time each plate separately to ensure accurate incubation times, follow protocol. Maintain the lab room temperature within 20º 25ºC (68º 77ºF). Do not run assays under air conditioning vents or near cold windows. Make sure plates and reagents are brought up to room temperature. Keep the kit components out of the kit box for at least 1 hour before starting the assay. Make sure the wavelength is 450 nm for the assay and read the plate again. Verify reader calibration and lamp alignment. Check records to see how many times the kit has cycled from the refrigerator. Check to see if the kit was left at extreme temperatures for too long. If the protein level in the diluted sample is low use a higher cell number (i.e. grow cells in larger wells) to obtain a higher reading. If the cell number is low use a higher cell number (i.e. grow cells in larger wells) to obtain a higher reading. BIOO RESEARCH PRODUCTS 5

8 High Background or High Optical Density (OD) Readings Possible Causes Recommended Action Poor quality water was used in wash solution. Substrate solution has deteriorated. There was insufficient washing or poor washing technique. Reader was malfunctioning or not blanked properly. This is a high possibility if the OD readings were high and the color was light. Lab temperature was too high Reagents were intermixed, contaminated or prepared incorrectly. Cross-reactivity between the user supplied antibody and the secondary antibody in the kit High Intra-Plate or Inter-Plate Variance Possible Causes Recommended Action Multichannel pipette was not functioning properly. There was insufficient washing or poor washing technique. Inconsistent incubation times occurred from plate to plate. Pipette was working improperly. Kit plates, reagents and samples were at different temperatures. Reagents used were intermixed from different kit lots, or the kits were of different expiration dates. If water quality is questionable, try substituting an alternate distilled water source to prepare the wash solution. Make sure the substrate is colorless prior to addition to the plate. Use the number of washes per the protocol instruction. Make sure that 250 µl of wash solution is dispensed per well per wash. Verify the performance of the pipettors or multichannel pipettor; have the pipettors repaired if any ports drip, dispense or aspirate poorly. Verify the reader s performance using a calibration plate and check the lamp alignment. Verify the blanking procedure, if applicable, and reblank. Maintain the room temperature within 20º 25ºC (68º 77ºF). Avoid running assays near heat sources or in direct sunlight. Ensure that the correct reagents were used, that working solutions were prepared correctly and that contamination has not occurred. Run a negative control using no primary antibody. This should yield a minimal amount of signal. Verify pipette calibration and check that tips are on tight. Be sure all channels of the pipette draw and dispense equal volumes. Use the number of washes per the protocol instruction. Make sure that 250 µl of wash solution is dispensed per well per wash. Verify the performance of the pipettors or multichannel pipettor; have the pipettors repaired if any ports drip, dispense or aspirate poorly. Time each plate separately to ensure consistent incubation times. Check the pipette calibration. Verify that pipette tips are on tight before use and that all channels draw and dispense equal volumes. Make sure to allow sufficient time for kit plates, reagents, standards and samples come to room temperature (20 25ºC/68 77ºF). Larger volumes will require longer equilibration time. If using a water bath to hasten equilibration, make sure it is maintained at room temperature; do not use a warm water bath to warm reagents, samples and kit standards. Carefully label each user-prepared reagent to make sure the reagents are not intermixed. Kits with different expiration dates might generate different range of OD readings; however, the relative absorbance values will typically be comparable. Bioo Scientific Corporation 3913 Todd Lane Suite 312 Austin, TX USA Tel: Fax: (512) Made in USA BIOO Research Products Group info@biooscientific.com techsupport3@biooscientific.com BIOO RESEARCH PRODUCTS 6