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