Prestige Antibody Protein Array Procedure
The binding specificity of the purified Prestige Antibodies® is determined on protein arrays, using PrEST protein fragments, to ensure high specificity toward its antigen and low background binding.
PBS (P5368) – 8.1 mM Na2HPO4 (S3264), 1.5 mM KH2PO4 (P9791), 137 mM NaCl (S3014), and 2.7 mM KCl (P9541), pH 7.4
PBST – PBS with 0.1% (v/v) TWEEN® 20 (P3563)
Blocking Buffer – PBST with 3% bovine serum albumin (A7888)
All washes are performed at room temperature (RT) on a shaker.
Antibody dilution with PBST:
- for antibody concentrations £0.04 mg/ml, 1:500
- for antibody concentrations ³0.05 mg/ml, 1:3,000
Note: The specified working dilutions of the antibodies are to be considered as guidelines only. Optimal dilutions must be determined by the user.
- The PrEST protein (the antigen) is diluted 1:10 in PBS containing BSA (100 mg/ml) and printed with 383 other PrEST antigens on epoxy coated microarray slides.
- The slides are incubated overnight at 37 °C.
- Unreacted epoxide groups are blocked by incubating the printed slide in Blocking Buffer for 60 minutes.
- 60 ml of the primary antibody solution are applied to each well and the slide is incubated for 60 minutes. The arrays are separated by a silicon mask.
- The slide is washed twice, each time for 5 minutes with PBST followed by a third wash with PBS for 5 minutes.
- The secondary antibody solution is prepared by diluting the goat anti-rabbit IgG – Alexa Fluor® 647 conjugate 60,000-fold in PBST.
Note: The secondary antibody is fluorescently labeled, and thus, light sensitive. The slide should be kept in the dark during the rest of the procedure.
- The secondary antibody solution is added to the slide, which is incubated for 60 minutes in the dark.
- The slide is washed twice, each time for 5 minutes with PBST followed by a third wash with PBS for 5 minutes in the dark.
- The slide is spin dried for 10–15 seconds using a table centrifuge prior to scanning.
Prestige Antibodies is a registered trademark of Sigma- Aldrich® Biotechnology LP and Sigma-Aldrich Co.
Alexa Fluor is a registered trademark of Molecular Probes.
TWEEN is a registered trademark of Croda International PLC.