After transfer, to prevent the non-specific binding of the primary and secondary antibodies, membrane blocking was performed in a blocking solution consisting of 5% non-fat dried skimmed milk dissolved in TBST buffer [Tris-buffered saline- Tween 20 (10 mM Tris base, 100 mM NaCl and 0.1% Tween 20, pH 7.6)].
Following SDS PAGE, proteins were transferred to a nitrocellulose membrane, blocked and incubated with a goat polyclone anti-GFP (AbD Serotec).
Doubling dilutions of the antigen were run on an SDS-PAGE gel and transferred to a PVDF membrane, blocked and then probed with a single dilution of crude bacterial cell extract from an overnight induction.
Proteins were transferred to a nitrocellulose membrane, blocked, and immunolabeled overnight at 4°C with a primary antibody (detailed in Additional file 1).
Protein extracts were run on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane, blocked in 3% non-fat milk powder in PBS for 1 h and probed with anti-c-MYC antibody.
The proteins were transferred onto a PVDF membrane, blocked with 5% nonfat milk for 1 h at room temperature, and probed with a primary antibody (1∶1000) overnight at 4°C.
Chlamydial proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane, blocked, and washed according to standard procedures [24].
The gel was electro-transferred to a nitrocellulose membrane, blocked in 5% milk, and probed with anti-AR or anti-GAPDH antibodies overnight.
The chlamydial proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane, blocked, and washed according to standard procedures [31].
The extracted proteins equivalent to 1×105 5×10505 cells were loaded and separated in 15% SDS-PAGE gel, transferred to a PVDF membrane, blocked with 5% milk in TBS-T buffer and blotted with each antibody at recommended dilution by manufactures.
