2D第一相是IEF玻璃管的请进
丁香园论坛
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刚开始做,希望多认识几个朋友,
做了好几次,都没有出来结果,请多指点
Gel Recipe
6.6 g Urea
1.59 ml Acrylamide/Bis (28.38g:1.62g to 100ml)
2.4 ml 10% Triton X-100
0.48 ml ampholyte pH5-8
0.12 ml Ampholyte pH3-10 (optional)
2.4 ml Water
12 μl TEMED
90 μl 3% Ammonium Persulfate
This makes 12 ml total volume, enough to cast two pieces of slab gel (OCEAN mini gel system). Degas the solution before addition of ammonium persulfate.
Sample buffer (2X)
9.5 M urea 5.7 g
2.0% Triton X-100 2.0 ml 10% Triton X-100
5% 2-Mercaptoethanol 0.5 ml
1.6% Ampholyte (pH 5-8) 400 μl
0.4% Ampholyte (pH 3-10) 100 μl
Bromophenol blue 0.5 mg (optional)
Dilute to 10 ml with water. Warm in water bath to dissolve urea. Aliquot and store in freezer.
Sample Preparation
Mix equal volume of sample and sample buffer. Incubate at room temperature for 10 - 15 min.
Running Buffers
Upper chamber (-): 20 mM NaOH
Lower Chamber (+): 10 mM H3PO4
Pre-electrophoresis (optional)
200 V of 10 min, 300 V for 15 min, and 400 V for 15 min. Change the running buffer before loading.
Sample loading
Wash the wells with running buffer. Overlay the wells with sample overlay buffer (dilution of the sample buffer with 2 vol. of water). Load sample to the bottom of the wells.
Electrophoresis
300 V for 10 min, 500 V for 15 min, and 750 V for no less than 3.5 hours.
Staining
Fix in fixing solution (10% acetic acid/50% methanol) for 30-60 min with one change of the solution. Then stain as for SDS gel.
做了好几次,都没有出来结果,请多指点
Gel Recipe
6.6 g Urea
1.59 ml Acrylamide/Bis (28.38g:1.62g to 100ml)
2.4 ml 10% Triton X-100
0.48 ml ampholyte pH5-8
0.12 ml Ampholyte pH3-10 (optional)
2.4 ml Water
12 μl TEMED
90 μl 3% Ammonium Persulfate
This makes 12 ml total volume, enough to cast two pieces of slab gel (OCEAN mini gel system). Degas the solution before addition of ammonium persulfate.
Sample buffer (2X)
9.5 M urea 5.7 g
2.0% Triton X-100 2.0 ml 10% Triton X-100
5% 2-Mercaptoethanol 0.5 ml
1.6% Ampholyte (pH 5-8) 400 μl
0.4% Ampholyte (pH 3-10) 100 μl
Bromophenol blue 0.5 mg (optional)
Dilute to 10 ml with water. Warm in water bath to dissolve urea. Aliquot and store in freezer.
Sample Preparation
Mix equal volume of sample and sample buffer. Incubate at room temperature for 10 - 15 min.
Running Buffers
Upper chamber (-): 20 mM NaOH
Lower Chamber (+): 10 mM H3PO4
Pre-electrophoresis (optional)
200 V of 10 min, 300 V for 15 min, and 400 V for 15 min. Change the running buffer before loading.
Sample loading
Wash the wells with running buffer. Overlay the wells with sample overlay buffer (dilution of the sample buffer with 2 vol. of water). Load sample to the bottom of the wells.
Electrophoresis
300 V for 10 min, 500 V for 15 min, and 750 V for no less than 3.5 hours.
Staining
Fix in fixing solution (10% acetic acid/50% methanol) for 30-60 min with one change of the solution. Then stain as for SDS gel.
