"
Team:Hannover/Protocols/Detection/Proteins
From 2014.igem.org
Protocols / Detection of Proteins
Material: |
|
| Coomassie-blue R250 | 0.05 % (w/v) coomassie R250, 25 % (v/v) isopropanol, 10 % (v/v) glacial acetic acid |
| Semi-Dry Blotting device | |
| Whatman paper | |
| Seesaw | |
| Blotting buffer | 250 mM Tris-HCl; pH 8.3, 1.92 mM glycine |
| Blocking Solution | |
| 10 x PBS buffer | 2.56 g NaH2Po4 x H2O, 14.90 g NaHPO4 x 2 H2O, 87.66 g NaCl, ad 1 l H2O; pH 7.4 |
| Antibodies | |
| Substrate buffer | 0.5 mM MgCl2, 100 mM Tris-HCl; pH 9.2, for the enzymatic staining add: 1 % (v/v) BCIP,1 % (v/v) NBT |
Protocol
After proving the technical quality of the gel run, the SDS gels were stained in Coomassie blue R250 for 30 min first. After the complete gel was penetrated by the dye, arginine related bindings were uncovered by washing with H2O on a slow moving seesaw overnight.
To specifically detect the produced target protein, the separated proteins obtained by the SDS-PAGE were transferred onto a PVDF membrane electrically by the PerfectBlue Semi-Dry Electro Blotter (PEQLAB Biotechnologie GmbH, Erlangen) then. Therefore, the gel was placed upon the membrane, which was centered between three sheets of Whatman paper beneath and four on top of it. For better flow of electrical current, the Whatman papers were immersed in 1 x blotting buffer and covered the membrane liberally. After removing the air bubbles, blotting was carried out for 48 min at 20 V, at a current limit of 0.5 mA cm-1. To make sure that proteins were transferred to the membrane, both a coomassie staining with the blotted gel and a Ponceau staining with the membrane were performed. For the latter, gels were penetrated by Ponceau solution for 10 min and washed with H2O for an additional 10 min, to remove membrane-related bindings again. Once it was verified that proteins were transferred, the membrane was placed in 40 ml 1 x RotiBlock© blocking solution overnight. Before the membrane was incubated in the antibody dilutions, a single washing with 1 x PBS buffer was done. After incubating both the primary and the secondary antibody, the membrane was washed with 1 x PBS buffer twice, once by rinsing the membrane for a few seconds, and once by cleaning it properly for 15 min on a slow moving seesaw. After the last wash following the secondary antibody incubation, the membrane was accustomed to the substrate buffer by incubation for 5 min. Finally, the membrane was placed in a second 1 x substrate buffer including 1 % (v/v) of NBT and BCIP, which were converted by the antibodies conjugated alkaline phosphatase. Depending on the specificity of the primary antibody, the membrane´s incubation time in the substrate buffer varied. The substrate conversion times are stated in the figure descriptions.
