Mini SDS-PAGE
1) Fix glasses (80 x 60 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 2 gels)
(17%)
Acrylamide for separating gel 9.7 ml
Separating gel buffer (pH 6.8) 6.3 ml
Water (MQ) 0.8 ml
10% APS 120 μl
TEMED 20 μl
Mix the solutions and fill the plates about 1.5 cm from top.
Caution: Pour the solutions into the plates immediately after adding 10% APS and TEMED.
3) Overlay the separating gel solution with 0.5 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 2 gels)
(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 μl BPB solution.
12) Run the gel at 20 mA until the BPB line reaches about 5 mm near the bottom.
13) Disconnect electricity, unfix clips remove plates.
14) Separate two plates with a spatula.
15) Separate the stacking gel and take the separating gel. This separating gel will be used for blotting.
Blotting
1) Wash PVDF membrane in methanol for 5 sec., transfer the membrane in 100 ml blotting solution C, shake for 5 min.
2) Wet 2 blotting papers (3 mm) in each A, B, and C blotting solution.
3) Place the separating gel in 100 ml blotting solution C and shake for 5 min.
(If the is kept in blotting C for longer time, the gel will be enlarged)
4) Wet the semidry blotting apparatus with water (MQ).
Place the blotting papers A on the blotting plate then B (earlier kept in blotting solution A and B)
carefully avoiding air, then place PVDF membrane, the gel then blotting paper C (earlier kept in blotting solution C).
5) Connect power supply (upper - and lower +). Run the blotting at 40 mA for 90 min for each mini gel.
6) After blotting wash the membrane in 100 ml water (MQ).
7) Stain the gel for 1 to 2 min in Ponceau 3S staining solution.
8) Destain the gel in 1% acetic acid until the background is lightened (clear).
9) Wash the gel with water (MQ) for 1 to 2 min.
10) Air dry the PVDF membrane and preserve in vinyl packet.
Cut the protein bands for analysis by a protein sequencer. (Fig.2)