R&D Systems ELISA for Human VEGFA

Judith Sunzenauer (last update 11.6.2013)

Procedures

Preparation:

• Bring all reagents and samples to room temperature before use.
• Run duplicates.
• Samples: addditional centrifugation step at 10.000xg for 10min at 4°C is necessary. Dilute samples 1:2 with Calibrator Diluent RD6U.
• Prepare Wash Buffer: Add 20ml of Wash Buffer Concentrate to 480ml a.dest (1:25 Dilution)
• Discard Calibrator Diluent RD5K (only for cell culture supernate samples!!!)
• Reconstitute VEGF Standard: add 1ml Calibrator Diluent RD6U to 1vial of VEGF Standard (concentration of stock: 2000pg/ml). Allow the standard to sit for a minimum of 15min prior to making dilutions.
• Produce the dilution series for VEGF Standardcurve:

Prepare 8 prelabeled (form #1-#8) polypropylene tubes and fill each tube with 500µl of Calibrator Diluent RD6U. Transfer 500µl Standard from the original vial to tube #1, mix thoroughly and transfer 500µl from tube #1 to #2,….. continue until tube #7. Fill tube #8 with Calibrator Diluent RD6U.

Assay Procedure:

• Add 100µl of Assay Diluent RD1W to each well.
• Add 100µl of Standard Dilution #1-8 or sample per well.
• Seal the Plate and incubate 2h at room temperature.

• Aspirate each well.
• Wash 3 times with 400µl Wash Buffer.
• After the last wash, remove any remaining liquid and invert the plate and blot it against clean paper towels.
• Add 200µl of VEGF Conjugate to each well.
• Seal and incubate for 2h at RT.

Prepare Substrate Solution: mix color Reagents A and B in equal volumes within 15min of use. Protect from light!

• Wash 3 times with 400µl Wash Buffer.
• Add 200µl of Substrate Solution to each well.
• Incubate for 25min at RT and protect from light.

• Add 50µl Stop Solution to each well. Gently tap the plate to ensure mixing.
• Determine the OD at 450nm within 30min. Set wavelength correction to 540nm or 570nm.

Calculation of Results:

• If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
• Concentrations of the Standard Dilutions:

Sensitivity:

Handling Photometer + Software:

Make sure the laser is warmed up prior to scanning for 15min

Materials