Youssef Farhat, Written 5/3/13, Last updated 5/6/14
Download the PDF or Microsoft Word versions of this protocol
Introduction
PAI-1 is a protein that plays an important role in blood clotting, wound healing and possibly even scar tissue formation. Thus, there is a need to understand how its expression and protein levels are regulated in a number of cells. This Mouse Active PAI-1 ELISA kit is a Sandwich ELISA that is useful for exclusively evaluating the protein levels of Active Mouse PAI-1 (excluding the inactive and latent forms of PAI-1) in samples from mice or mouse cells (this kit may not work with other species).
Materials
- Mouse PAI-1 Activity Assay (Molecular Innovations, #MPAIKT). This kit includes the following
- 96-well strip plate (pre-coated with uPA and blocked)
- 10X Wash Buffer
- Mouse Active PAI-1 Standard
- Anti-Mouse PAI-1 Primary Antibody
- Anti-Rabbit Secondary Antibody (HRP-conjugated)
- TMB Substrate Solution
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- Bovine Serum Albumin, Fraction V, protease free (Roche, #03117332001)
- Stock Solutions
- Stop Solution (2 N H2SO4 [R&D Systems, #DY994])
- Typical lab equipment and supplies, such as a horizontal plate shaker (capable of 300 rpm), a plate reader, 50-mL tubes, stir bars, stir plate, pipettors, pipette tips, beakers, distilled water, 1.5 mL Eppendorf tubes, paper towels, plastic wrap (for sealing the plate), reagent reservoirs (for multi-channel micropipettes)
Procedure
This ELISA kit takes about three hours to perform once the stock solutions have been prepared. As always, samples should be thawed out if they were previously frozen and kept on ice until adding to the ELISA plate. Before performing the assay, all materials should be brought to room temperature.
1) Reagent Preparation (Estimated time: 15 min)
- Prepare 40 mL of Tris-Buffered Saline (TBS) by combining the following:
- 4 mL of 1.0 M Tris-HCl (pH 7.4) Stock Solution
- 3 mL of 2.0 M NaCl Stock Solution
- 33 mL of deionized water
- This solution has a final composition of 100 mM Tris-HCl and 150 mM NaCl
- Prepare 33 mL of Block Buffer by combining the following in a beaker:
- 1 g BSA
- 33 mL of TBS
- Mix with a stir rod until completely dissolved (this takes about 5 min)
- Prepare the Wash Buffer by combining the following in a clean, 500-mL bottle:
- 50 mL of 10X Wash Buffer
- 450 mL of deionized water
2) Perform the Assay (Estimated time: 2-3 hours)
- Prepare the PAI-1 protein standard:
- Add 5 mL of Block Buffer to the vial containing 250 ng of lyophilized Mouse Active PAI-1 Standard and mix thoroughly (concentration becomes 50 ng/mL).
- In a set of cDNA strip tubes (kept on ice), serially dilute the Mouse Active PAI-1 Standard 1:2 (vol:vol) in Block Buffer, leaving the last (8th tube) blank (Pure Block Buffer, so that it serves as the Assay Blank).
- i. Place 210 µL of Active PAI-1 Standard (50 ng/mL) in the first tube.
- ii. Place 210 µL of block buffer in the last seven tubes.
- iii. Serially dilute another 105 µL of Active PAI-1 Standard into the next 6 strip tubes, leaving the last tube unmixed (Pure Block Buffer) to serve as the Assay Blank.
- If necessary, dilute samples in Block Buffer to bring them within the working range of the assay (0.069-50 ng/mL).
- Add 100 µL of the samples and standards to the plate according to your plate layout (Click here to download our 96-well Plate Layout print-outs).
- Seal the plate and incubate it for 30 min at room temperature on a horizontal plate shaker set to 300 rpm.
30 minutes later…
- Prepare the Primary Antibody:
- Add 10 mL of Block Buffer to the vial containing the anti-PAI-1 antibody and mix thoroughly.
- Wash the wells three times with 400 µL of Wash Buffer.
- Add 100 µL of Primary Antibody to each well of the ELISA plate
- Seal the plate and incubate it for 30 min at room temperature on a horizontal plate shaker set to 300 rpm.
30 minutes later…
- Prepare the Secondary Antibody:
- Briefly centrifuge the vial containing Secondary Antibody with a benchtop centrifuge so that the droplet settles to the bottom of the tube.
- Combine 1 µL of Secondary Antibody and 10 mL of Block Buffer into a reagent reservoir.
- Wash the wells three times with 400 µL of Wash Buffer.
- Add 100 µL of Secondary Antibody to each well of the ELISA plate
- Seal the plate and incubate it for 30 min at room temperature on a horizontal plate shaker set to 300 rpm.
30 minutes later…
- Pour the Substrate Solution (~10 mL) into a clearly labeled reagent reservoir.
- Place 5 mL of Stop Solution into another clearly labeled reagent reservoir.
- Set up the plate-reader to shake the plate at medium setting for 15 seconds and then measure absorbance at 450 nm.
- Wash the wells three times with 400 µL of Wash Buffer.
- Add 100 µL of Substrate Solution to each well of the ELISA plate
- Incubate the plate for 2-10 min at room temperature, monitoring it closely for color change.
- At the desired time, add 50 µL of Stop Solution to each well of the ELISA plate.
- The best time to add Stop Solution is when the most dilute samples on your standard curve have produced a little bit of color.
- Read absorbance at 450 nm in the plate reader.
Citing this Protocol
Farhat, Y. “Total PAI-1 ELISA Protocol (Molecular Innovations).” Protocol Place. May 2013. Accessed on mm/dd/yyyy. <http://protocol-place.com/>
