Bitan:alisa

This is an ALISA protocol for protein concentration.

=Ingredients=

Buffers
PBS PBST 0.1% Tween_20

Coating Buffer
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*

Blocking Buffer
2% Amersham ECL Advance Blocking Reagent in PBST

Phosphate/Citrate Buffer
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*

Other Reagents
Monoclonal 6E10 iQ SYBR Green Supermix =Recipe=

Coating

 * 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

Block

 * 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

Aptamer Incubation

 * 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

Aptamer Recovery

 * 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.

Perform qPCR

Analysis

 * 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.

=References= A related protocol has been published: Yoshida Anal Bioanal Chem 2009