Yu:Western: Difference between revisions
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===Developing=== | ===Developing=== | ||
===Stripping=== | |||
====Stripping Buffer==== | |||
{| border="1" cellpadding="10" cellspacing="0" | |||
|width="300px" valign="top"|'''Solution''' | |||
|width="100px" valign="top"|'''Volume''' | |||
|- | |||
|1.5M Tris pH 6.8 | |||
|2.08 mL | |||
|- | |||
|β-Mercaptoethanol | |||
|0.5 mL | |||
|- | |||
|10% SDS | |||
|10 mL | |||
|- | |||
|milliQ H<sub>2</sub>O | |||
|37.42 mL | |||
|- | |||
|colspan="2"|Final Volume is 50 mL | |||
|} | |||
'''Procedure''' | |||
#Wash Membrane for 15-20 min in 1x [[Yu:PBS#PBST|PBST]] at room temperature to wash away developing solution | |||
#Incubate membrane in 50 mL of stripping buffer on a shaking platform for 30 min at 50°C | |||
#Wash Membrane for 15-20 min in 1x [[Yu:PBS#PBST|PBST]] at room temperature | |||
#Block and reprobe membrane with desired antibody |
Revision as of 11:07, 11 September 2009
Western Blotting
SDS PAGE
1.Cast SDS-PAGE gel and assemble the gel apparatus. |
2. Add 10μL of desired sample and 5μL 3x Gel loading buffer to an eppendorf tube and boil for 5 minutes. Do this in separate tubes for each sample to be analyzed. After boiling, samples may be kept at room temperature until loaded |
3. Load 12μL to 15μL of sample into each lane. Load protein prestain marker, as well as BenchMark, if desired. Load any empty lanes with 1x Gel loading buffer |
4. Run gel until the loading buffer is just running off the bottom of the gel |
Transfer
Immunoblotting
Developing
Stripping
Stripping Buffer
Solution | Volume |
1.5M Tris pH 6.8 | 2.08 mL |
β-Mercaptoethanol | 0.5 mL |
10% SDS | 10 mL |
milliQ H2O | 37.42 mL |
Final Volume is 50 mL |
Procedure