Protein biochemistry

shaking at 37°C for 2 hours. The buffer was replace d by 40 ml DIG II buffer containing an AP-conjugated antibody against digoxigenin in a 1:10,000 dilution and the membrane was incubated shrink-wrapped in this buffer at 37°C for 45 min. This was followed by two more 15 min washing steps in DIG washing buffer and an equilibration for 5 min in DIG III at RT.

Each side of the membrane was then soaked for 2.5 min in DIG III buffer containing CSPD in a 1:100 dilution and the membrane was placed between two plastic foils in a radiographic cassette and, after pre-incubation for 10 min at 37°C, exposed to X-ray films for 15-120 min at 37°C.

3.4.3 Affinity purification of recombinant proteins

3.4.3.1 Purification of GST-tag fusion proteins

Glutathione Uniflow resin was provided as a 50% (v/v) solution. 400 µl of the resin was used per 100 ml of original E. coli culture expressing recombinant proteins. The resin was washed twice with 10 ml PBS and centrifuged at 500 x g for 2 min in between each wash step. The protein-containing supernatant was added to the washed resin and incubated rotating at 4°C for 1 hour to enable binding of GST -tag fusion proteins to the resin. The protein-bound resin was subsequently sedimented from the mixture by centrifugation at 500 x g and 4°C for 2 min and the supernatant was r emoved. The resin was washed 4 times with 5 ml ice-cold PBS. The solution was incubated rotating at 4°C for 10 min between each wash step and sedimentation was achieved by centrifugation at 4°C and 500 x g for 2 min. The supernatant was removed in each case with the help of a 1 ml syringe and a 23 gauge needle to minimise loss of resin. The recombinant protein was eluted after washing by incubating the resin rotating for 10 min at 4°C in 200 µl glutathione elution buffer and centrifugation at 500 x g and 4°C for 2 min. The supernatants containing the purified, eluted GST-tag fusion proteins were stored for a maximum of 3 days on ice at 4°C until further use.

Because of the poor expression levels experienced for LmxMPK6 and GST-LmxMPK6K33M those proteins were expressed using 200 ml E. coli cultures. The cell suspensions were distributed to two 15 ml centrifugation tubes for the preparation of cell lysates but were then incubated with the same amount of Glutathione Uniflow Resin as other proteins from 100 ml cultures and subsequently treated exactly the same.

3.4.3.2 Purification of His-tag fusion proteins

Per 100 ml of original E. coli protein expression culture 400 µl of Chelating Sepharose Fast Flow were loaded with Co²⁺. To achieve this, the sepharose was initially sedimented by centrifugation at 500 x g and 4°C for 2 min to f acilitate the removal of the storage solution. The sepharose was subsequently washed twice with 1 ml H2O, once with 200 µl 0.1 M CoCl2 solution, once again three times with 1 ml H2O and finally with 200 µl His-purification binding buffer. Between each washing step, the solutions were rotated at RT for 5 min, the sepharose was sedimented by centrifugation for 2 min at 500 x g and 4°C and the supernatant was removed with the help of a 1 ml syringe with a 23 gauge needle.

The resulting Co²⁺-loaded sepharose was mixed with the protein supernatant from step 3.4.2 and with 5 ml His-purification binding buffer and incubated rotating for 1 hour at 4°C.

The sepharose was subsequently washed one time each with 6 ml His-purification washing buffer, 1 ml His-purification binding buffer and 1 ml His-purification washing

buffer. The solution was rotated at 4°C for 10 min between each washing step and each time sedimented by centrifugation at 500 x g and 4°C for 2 min. The purified protein was not eluted but remained bound to the resin and was stored on ice at 4°C in 200 µl His-purification washing buffer.

3.4.3.3 Purification of S-tag fusion proteins

The preparation of lysates for S-tag protein purification differed slightly from the method described in 3.4.2 as the harvested and washed cells were suspended in 10 ml S-tag purification binding/washing (B/W) buffer and afterwards incubated with 1 ml of 10% (v/v) Triton X-100. All other steps proceeded according to the protocol described above. The protein supernatants (3.4.2) were incubated with 200 µl S-protein agarose slurry rotating at RT for 45 min. The protein-bound resin was sedimented by centrifugation at 500 x g for 5 min and washed 5 times with 5 ml B/W buffer, interspersed with 10 min incubation on the roller at RT. The proteins were not eluted from the resin as this would require the addition of 3 M MgCl2 and a subsequent dialysis against B/W buffer to remove MgCl2. All this posed a high risk of protein loss by precipitation, which is why the protein was kept bound to the resin and was stored on ice at 4°C in 200 µl B/W buffer.

3.4.4 Thrombin cleavage of GST-tag fusion proteins

250 µg of GST-tag fusion protein was incubated over night at 20°C with 0.5 U thrombin.

3.4.5 Determination of protein concentration by Bradford assay

500 µl Bradford reagent was mixed with 10 µl protein sample and incubated for 2-3 min.

The protein concentration was measured with the help of an Eppendorf BioPhotometer in the calibrated Bradford mode.

3.4.6 Discontinuous SDS polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PA gels were prepared at RT and consisted of 4% stacking gels and varying resolving gels of 8-15% concentrations. Protein samples were mixed with ¼ volume of 5 x SDS-PAGE sample buffer and incubated at 95°C fo r 10 min to denature proteins. After 5 min on ice a maximum of 30 µl per sample was loaded in the gel pockets. The proteins were separated at 20 mA while in the stacking gel and at 30 mA while in the resolving gel.

If a double SDS-PAGE chamber was used the applied electrical current was doubled.

Proteins of kinase assays were separated at 15 mA in the stacking gel and at 20 mA in the resolving gel to achieve a better separation of protein bands. A pre-stained NEB

protein molecular weight marker was used to estimate the molecular weight of separated proteins.

3.4.7 Staining of SDS-PA gels

3.4.7.1 Coomassie staining

After completion of gel electrophoresis the gels were incubated shaking at RT for approximately 1 h, but at least 30 min in Coomassie staining solution. The gels were subsequently washed several times with Coomassie destaining solution until the protein bands were distinctly visible and the blue background was sufficiently reduced. The gels were either dried or stored in ddH2O.

3.4.7.2 Silver staining

The gel was incubated for 30 min in silver staining fixing solution and subsequently washed thrice with washing solution I for 10 min each. All steps took place on a benchtop shaker at RT. The gel was pre-treated with solution I for 5 min after which it was washed twice with ddH2O for 1 min. Incubation with solution II took place for 10 min followed by two washing steps in ddH2O for 1 min and the incubation in developing solution. When the gel bands were distinctly visible the developing solution was discarded and the reaction was brought to an end by the addition of stopping solution. Finally the gel was washed with washing solution II and III for 30 min each and left in storage solution until it was dried.

3.4.8 Drying of PA gels

Stained gels were incubated in gel drying solution for at least 30 min. Sheets of cellophane were soaked in water and the pre-incubated gels were placed between two cellophane sheets which were mounted within gel drying frames. The absence of air bubbles was crucial to dry gels without tearing. The construction was placed in a fume hood for 12-24 hours.

3.4.9 Immunoblot analysis

Proteins that had been separated by SDS-PAGE were transferred to an Immobilon-P PVDF membrane by semi-dry electroblotting. The stacking gel was removed from the resolving gel, which was then placed in transfer buffer. Eight Whatman cellulose blotting papers were cut to the same size as the resolving gel and soaked in transfer buffer. An Immobilon-P PVDF membrane of the same size was incubated 1 min in 100 % methanol and 10 min in transfer buffer. The blot was set up starting at the anode. Four Whatman

papers were stacked and the membrane was carefully and without inclusion of any air bubbles placed on top of the stack. The SDS-PA gel was positioned on top of the membrane under a stack of 4 Whatman papers. A plastic pipette was carefully rolled over the set-up to remove any remaining air bubbles. The protein transfer took place at 4 mA/cm² for 30 min, if one gel was blotted, or 45 min, if two gels were blotted.

The SDS PA gel was Coomassie-stained after completion of electroblotting and the membrane containing the transferred proteins was blocked by shaking in Immunoblot blocking solution for 1 hour at 37°C. The incubatio n with primary antibody was conducted by subsequently placing the membrane in a 50 ml centrifugation tube, which included an appropriate dilution of antibody in blocking solution, and rotating the tube over night at 4°C. The membrane was then washed four times with P BST (TBST in the case of 4G10) by shaking for 5 min at RT. The second antibody was also diluted in blocking solution and incubated with the membrane for 1 h at 37°C.This wa s followed by 3 washing steps in PBST (TBST) and 2 washing steps in PBS (TBS), lasting 5 min each. Detection of protein bands was achieved by soaking the membrane with equal measures of the two solutions of the SuperSignal West Pico Chemiluminescent Substrate Kit. The membrane was placed between two plastic foils in a radiographic cassette and X-ray films were exposed for 1 s to 30 min.

3.4.10 Stripping-off antibodies from an immunoblot

To be able to re-probe an immunoblot with a different antibody, the previous one had to be removed. This was achieved by incubating the membrane for 30 min in a container that was filled with 100 ml immunoblot stripping solution and that was placed in a 65°C hot water bath. The membrane was subsequently washed twice for 10 min with PBST or TBST before it was blocked with fresh blocking solution. The detection of protein bands using a different antibody could follow.