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Procedure:
SDS-PAGE General Features:
Apparatus Bio-Rad (Hoefer)
Spacers:.75 mm
Comb: 10 wells/comb
Running conditions:
Adding:150 volts 601
Runnig:500 volts 90-1201
Keep refrigerated in the cold room.
To Cast a Gel:
12% running gel 6% stacking gel
After the Chamber Assembly:
1. Prepare 1 ml of 22% acrylamide to seal the chamber bottom:
0.75 ml Acryl. stock
0.25 ml Run. buff.
" ul Amm. Pers. 40%
1 ul TEMED.
Pipette -immediately between the glasses and wait for a complete polymerization (about 51).
2. Prepare 20 ml of Running Gel:
8 ml Acryl. Stock
5 ml Running Gel Buff.
7 mlH20
30 ul Amm. Pers. 40%
15 ul TEMED
Fill the chamber just 1 cm below the comb. Fill the chamber completely using water and wait for gelling (about 15301).
3. Prepare Stacking Gel 10 ml:
" ml Acrylam. Stock
2.5 ml Stacking Gel Buffer
5.5 ml H20
30 ul Amm. Pers.
15 ul TEMED
Discard the water and put the comb in the chamber. Fill with the stacking gel solution and leave polymerizing 15-301.
Sample Preparation
1. Warm the sample buffer 100 in boiling water.
2. Prepare the samples with the correct amount of protein preparation and water.
3. Add, to the sample buffer, 10% of mercaptoethanol and mix
gently. Put the correct amount of sample buffer (SX) in each sample.
4. Boiling for 31.
5. Wash the wells, twice, with electrode buffer lx and load the samples.
6. Fill to the top of each well using electrode buffer. SDS Gel Treatment
1. Fixing solution:301
20% TCA -30% Methanol in H20
2. Staining-Solutions: 1 h
Sol A = 3% Coomassie G or R 250
900 ml Methanol
100 ml H20
Sol B = 10 9 CUS04(may be made without this substance)
800 Ml H20
00 ml Acetic acid (mix in equal volume Sol A and B 100 ml)(tinol volume)
3. Destaining Solution:
10 9 CUS04(may be made without this substance)
1300 ml H20
200 ml AEetic Acid
500 ml Methanol
Repeat many times with frequent changes of destaining 100 ml each)
WB Procedure
1. Put the transfer buffer in the cold room some hours before using.
2. For 1 hour transfer: electrical conditions 200 V -0.4 Amp (at the beginning).
For OIN transfer: electrical conditions 100 mamp - 30 V (at the beginning).
3. Treatment of membrane after transfer:
Cut the membrane exactly like the gel.
Put the membrane in the blocking solution:
3-t BSA
50 mM TrispH 7.4
150 mM NaCl
1 mM EDTA
Leave under shacking at 37oC for 2 hours. After you can use immediately or store f or a few days in the cold room or freezer in -200C for months.
Procedure for Immunoblot Hybrid:
1. If the membrane was frozen, leave to equilibrate at room temp. After wash 51 with washing solution:
Sol 1: 1% BSA
50 mM Tris
150 mM NaClpH 7.4
5 mM EDTA
0.05% NP40
2. Incubate your antibody, dilute in the incubation solution.
Sol 2: 3% BSA
50 mM Tris
150 mM NaClpH 7.4
1 mM EDTA
0.05% HP40
Leave at least 1 hour at room temperature
3. Wash 3x 51 with Sol. 1.
4. Incubate with the secondary antibody in Sol. 2 for 1 hour.
5. Wash 3x 101
6. Incubate with the substrate; this point can vary using different system.
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