Cleveland, Blotting, CBB Staining, Destaining, Drying

Cleveland Method 

 1) Fix glasses (160 x 140 x 1 mm) with clip keeping 1 mm space between the plates.
 2) Prepare separating gel solution in a 100 ml beaker with the following solutions and amounts
 
Solutions Amounts (for 1 gel)     (18%)
Acrylamide for separating gel    10.5 ml Separating gel buffer (pH 8.8)     6.3 ml 10% APS     120 µl TEMED      20 µl

Mix the solutions and fill the plates about 3 cm from top. Caution: Pour the solutions into the plates immediately after adding 10% APS and TEMED.  3) Overlay the separating gel solution with 1 ml water (MQ).  4) Leave the gel for 40 to 60 min at room temperature for polymerization.  5) Remove the overlaid water and pour the following stacking gel solution.  6) Prepare the stacking gel solution in a 100 ml beaker.


Solutions Amounts (for 1 gel)      (5%)
Acrylamide for stacking gel       1 ml Stacking gel buffer (pH 6.8)       3 ml Water (MQ)       2 ml 10% APS      30 µl TEMED      20 µl

Mix well and pour on the separating gel, insert comb. Caution: Pour the solutions into the plates immediately after adding 10% APS and TEMED.  7) Leave the gel for 20 min at room temperature for polymerization  8) Take out comb, clips and silicon tubes.  9) Clean the wells with a syringe. 10) Fix the gel plates with the apparatus (slab). Pour SDS-PAGE running buffer. 11) Dissolve the protein in 20 µl of SDS sample buffer (pH 6.8) and apply to a sample well in SDS-PAGE.    Overlay the sample with 20 µl of a solution containing 10 µl of Staphylococcus aureus V8 protease (Pierce, Rockford, IL, USA)    with 1 µg/µl in deionized water, and 10 µl of SDS sample buffer (pH 6.8). Add 30 µl BPB solution. 12) Run the gel until the sample and stack protease and sample in the upper gel and interrupted for 30 min to digest the protein. 12) Run the gel at 35 mA until the BPB line reaches about 5 mm near the bottom. 13) Disconnect the electricity, take out the plates. 14) Separate two plates with a spatula. 15) Separate the stacking gel and take the separating gel Blotting 1) Cut the PVDF membrane equal to the size of the gel. 2) Cut the whatman filter paper equal to the size of the membrane. 3) Wash PVDF membrane in methanol for a few seconds and transfer the membrane in 100 ml blotting solution C, shake for 5 min. 4) Wet two blotting papers (3 MM) in each A, B and C blotting solution. 5) Place the separating gel in 100 ml blotting solution C and shake for 5 min. 6) Wet the semidry blotting apparatus with water (MQ).    Place the blotting papers A on the blotting plate then B. Remove the air bubble if any.    Place PVDF membrane on it and then the gel and blotting paper C. 7) Connect power supply. Run the blotting at 140 mA for 90 min. 8) Wash the membrane in 100 ml water (MQ). 9) Stain the gel for 2-3 min in CBB stain. 10) Destain the gel in 60% methanol for 3 min twice. 11) Wash with MQ and air dry at room temperature.