I am conducting 2DE of hippocampal proteins. The protocol is as follows:
1. Sample preparation: hippocampus were solubilized in the lysis buffer (7M Urea, 2M Thiourea, 4% Chaps, 30mM Tris-cl, pH 8.5) and sonicated for 10s. Then the homogenates were centrifuged at 1,4000 rpm for 15min. The supernatants were added with ice-cold 100% TCA to a final concentration of 15% and incubated on ice overnight. Then the solution were centrifuged at 1,4000 rpm for 15min at degC to remove the supernatants. The precipitation were solubilized with three volume of ice-cold acetone. (Since the precipitants could not resolve in acetone, I have to sonicate them until the precipitants solubilize in acetone) Then the solution were centrifuged at 1,4000rpm for 15min at 4 degC. Repeat for three times. Then the precipitants were left on ice for 30min for the evaporation of acetone. Then the precipitant which is removed of acetone were solubilized in lysis buffer(7M Urea, 2M Thiourea, 4% Chaps, 30mM Tris-cl, pH 8.5) again.
2. IEF: The purified protein solution were added with rehydration buffer (8 M urea, 2 % CHAPS, 0.2% DTT, 2% (v/v) IPG buffer, pH 3–11 NL, 0.002% bromophenol) to the volume of 450μL. The IEF conditions: Strips were actively rehydrated at 20 degC for 18 h at 50 V, focused at a constant temperature of 20 degC beginning at step 300 V for 2 h, step 500 V for 2 h, step 1000 V for 2 h, gradiently 8000 V for 8 h, and finishing at 8000 V for 10 h step.
3. SDS-PAGE: After the IEF, the strips were equilibrated in 2% SDS, 30%Glycerol, 6M urea, 75mM Tris-cl 6.8, 0.002%Bromophenol blue with 1% DTT and subsequently 2.5 % IAA for 15 minutes, respectively. Then they were transferred to the 12.5% SDS-PAGE. The cathode and anode buffers were both the Tris-glycine-SDS (5X: 125mM Tris, 960mM Glycine,0.5% SDS, pH 8.3, what I used was 1X)buffer. 1W/Gel for 50min and 11W/ Gel for 6 hours.
The results showed heavy Horizontal streaking in the high molecule weight part and heavy vertical streaking in the basic part. I think it is due to the wrong sample preparation. I wonder if someone can suggest me the possible solution. Thank you for your help.
Attached is the new result of coomassie blue result.