Standard SDS-PAGE, CBB Staining, Destaining, Arabia Gum drying

Standard SDS-PAGE
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) (15%)
Acrylamide for separating gel 8.5 ml Separating gel buffer (pH 8.8) 6.3 ml Water (MQ) 2.0 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 8.8) 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) Boil the samples collected in SDS-Sample buffer at 95 ºC for 3 min. Add samples with a microsyringe or a pipette. Add 30 ¦Ěl BPB solution. 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, stain with CBB for 30-45 min. 16) Destain in destaining solution. Arabia Gum drying 1) Wash the destained gel with water MQ), Soak the gel in 20% Arabia Gum for 1 h. 2) Soak Cellophane in 10% glycerol, Place the cellophane in the Acryl plate. (Note: Make completely air free between the gel and Acryl plate). 3) Carefully put the gel on cellophane, apply a little 10% glycerol. (Note: Make completely air free between the gel and cellophane). 4) Cerefully put cellophane on the gel avoiding any air between the gel and cellophane. Put Acryl plate and fix with clips. 5) Place the Acryl plate with gel (vertical position) in a dry at 50 ºC for more than 8 h. 6) Remove the gel from the Acryl plate with a cutter.