For the immunological detection of proteins in biological samples we performed western blotting as a classical method (Towbin et al. 1992). In most cases proteins were overexpressed in 293HEK cells by transfection of plasmid DNA carrying the desired protein tagged with a polypeptide for better antibody recognition. Depending on the experiment the cells were lysed between 24 and 48 hours after transfection in different lysis buffers.
Cell lysis
Depending of the experiment and the protein fraction to be analyzed different lysis buffers were used. Lysis buffers differ in their ability to solubilize the proteins, the higher the sodium dodecyl sulfate (SDS) or other ionic detergents the higher the protein yield like RIPA buffer.
But these buffers denature proteins. For some applications and antibodies native
non-denatured proteins are needed. There lyse buffers without detergent or with relatively mild non-ionic detergents like Triton X-100 buffer have to be used.
Sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE)
Depending on the amino acids and their chemical character the charge of proteins vary and they have no linear relationship between weight and charge as DNA molecules have. To make a directed migration of proteins in an electric field possible SDS is used as a detergent and produces a negative charge on the protein surface. The tertiary and secondary structure of the proteins will be destroyed after the heat denaturation step. The polypeptide chains expose the hydrophobic regions and the lipophilic alkyls of the SDS bind via non-covalent bonds at the stoichiometry of 1 SDS per 2 amino acids. An additional treatment with dithiothreitol (DTT) reduces all intra- and intermolecular disulfide bonds.
The protein lysates were mixed with a 4x NuPAGE LDS (lithium dodecyl sulfate) sample loading buffer and DTT with a final concentration of 0.1 M. The samples were heated for 10 min at 70°C. 25-30 µg of protein lysate was loaded into the pockets of the NuPAGE Novex Bis- Tris gels (Invitrogen, life technologies). For the SDS-PAGE we used the XCell SureLock Mini-Cell chamber (Invitrogen) that was cooled by ice and the pre-cooled NuPAGE running buffer. The electrophoresis was performed at 200V for 30 min.
Transfer of proteins to membranes
According to the manufacturer’s protocol the transfer of proteins from a SDS-gel to a methanol activated PVDF membrane was performed with the XCell SureLock Western Blot System (Invitrogen) in transfer buffer containing 20% methanol. The transfer was performed at 30V for 2 hours and cooled by ice.
Detection of the proteins
After a successful transfer of the proteins the membrane was incubated in blocking buffer (5% non-fat milk powder in TBS-T for normal antibodies, 5% BSA in TBS-T for α-phospho antibodies) for 1 hour shaking at room temperature. Then the membrane was incubated with the primary antibody in a dilution 1:1000 in blocking buffer for 1-2 hours at room temperature or overnight at 4°C. After the incubation with the primary antibody the membrane was rinsed three times with TBS-T and washed four times in TBS-T shaking for 5 min at room temperature. Then the membrane was incubated with the secondary antibody that is conjugated with horseradish peroxidase (HRP) for 1 hour in a 1:5000 dilution with blocking buffer at room temperature. After a sufficient incubation the membrane was rinsed three times with TBS-T and washed seven times in TBS-T shaking for 7 min at room temperature. This last washing step is the most critical step to get low background signals.
For the detection the membrane was incubated for 1 min in enhanced chemiluminescence (ECL) detection solution mix (Perkin-Elmer) by gentle agitation at room temperature. Excess reagent was drained off and the membrane was placed into transparent plastic folders. The visualization of the signals was performed with the ChemoCam Imager (INTAS) a chemiluminescence scanner. The data were analyzed by the software (INTAS).
Co-Immunoprecipitation (Co-IP)
The immunoprecipitation is an often used method to precipitate a peptide-antigen out of a solution via a specific binding of the antigen to an antibody. A Co-IP is used to identify binding partners of that peptid-antigen. When the target protein has a strong binding with its interaction partner, the partner will be pooled out of the solution, too.
In brief, the cell lines were split onto 6-well dishes 18 h before transfection. On the next day the cells were transfected with the needed experiment setup, including the binding and interaction partners and binding controls. 24 hours after transfection the cells were washed with pre-cooled 1x PBS buffer one to two times. All following steps were done on ice to protect the proteins from degradation, especially the phosphorylated ones. Then 600µl of pre-cooled Triton-X cell-lysis-buffer were added to the cells. After the lysis step the samples were sonicated two times for ten seconds to break up the nuclei. To spin down the proteins and get rid of the cell debri a centrifugation step for ten minutes at 13.000 rpm and 4°C followed and the protein concentration was measured. Then the pre-washed and prepared Anti-Flag-M2 beads were added to the samples. The Anti-Flag-M2 beads contain a mouse antibody that recognizes every flag-tagged protein, binds on it and pulls it down. To bind the antibody coupled beads to the flag-tagged protein the samples have to incubate for two hours at 4°C in a rotation wheel. For very strong antibody antigen bonds the incubation is less.
Then the antigen-bound beads were purified by several washing and centrifugation steps. The more washing steps you make, the cleaner the western blot is. After the last washing step most of the lysis buffer was removed and the beads were dispensed in 60-70 µl of loading buffer containing DTT. Then the samples were heated for 10 minutes at 70°C, as protein denaturation step. The samples were load on SDS-gel depending on the protein amount and western blotting for protein identification followed.