This is an ALISA protocol for protein concentration.
Carbonate/bicarbonate (pH 9.6) 100 ml
0.293 g NaHCO3 (anhydrous)
0.159 g Na2CO3 (anhydrous)
- Use carbonate/bicarbonate to adjust pH to 9.6*
2% Amersham ECL Advance Blocking Reagent in PBST
50 mM citric acid and 100 mM sodium phosphate
0.74 g dibasic sodium phosphate (anhydrous)
1.3 g citric acid (anhydrous)
Dissolve in 50 ml water
*Adjust pH to 5.0*
*Add 40 μl fresh 30% H2O2 to 100 ml solution before use*
iQ SYBR Green Supermix
- Dilute 6E10 (1mg/ml stock) 1:1000 in Coating Buffer to make the Coating Solution
- Add 200ul Coating Solution to each well of a 96-well flat-bottom microtiter plate
- Incubate at room temperature with shaking for 2 hours or overnight in the cold room
- Dump Coating Solution into the sink
- Add 200ul Blocking Buffer
- Incubate at room temperature with shaking for at least two hours or overnight in the cold room
Incubation with protein preparation
- Prepare the desired dilution series of the protein from 500nM using PBS as diluent
- Add 100ul of each standard to the plate's wells in triplicate or quadruplicate
- Add 100ul of PBS in triplicate or quadruplicate as a control
- Incubate at room temperature for 1.5-2h
- Dilute detecting aptamer (100 μM) 1:1000 in Blocking Buffer
- Add 100ul of the aptamer solution to each well
- Incubate at room temperature with shaking for 1-1.5h
- Wash the plate with four 10min washes with 110ul PBST per well
- Tap the plate dry on a pad of Kimwipes
- Reverse the antibody-protein-aptamer sandwich by floating the microtiter plate in a 95 °C water bath and incubating for 10-15 minutes. This should irreversibly denature the capture antibody and target protein, the aptamer will be reversibly denatured.
- Transfer 1 μL of each well to a plate suitable for qPCR (conical wells coated with silicon to avoid non-specific interaction with protein).
- Add positive and negative PCR controls to the plate.
- Add primers to each well to a final concentration 100-500 nM.
- Add 12.5 μL iQ SYBR Green Supermix to each well.
- Add H2O to a final volume of 25 μL.
- Record the Ct values for all samples, standards and controls and compare to the positive control to estimate initial copy number.
- Interpolate samples into the standard curve to estimate protein concentration.
A related protocol has been published: Yoshida Anal Bioanal Chem 2009