Running SDS-PAGE: Difference between revisions
From Powers Wiki
(Created page with "Category:Protocols Category:Protein expression ==Running SDS-PAGE== #Make ready the samples to run #Make ready the sample running buffer #Make ready the gel #Have th...") |
No edit summary |
||
| (10 intermediate revisions by 4 users not shown) | |||
| Line 1: | Line 1: | ||
[[ | [[category:Gel]] | ||
[[category:Protein_Preparation]] | |||
[[Category:Protein expression]] | [[Category:Protein expression]] | ||
[[:File:SDS_PAGE.pdf|SDS PAGE]] | |||
==Running SDS-PAGE== | ==Running SDS-PAGE== | ||
| Line 11: | Line 14: | ||
#Place the stand with the gel in the SDS-PAGE apparatus bath | #Place the stand with the gel in the SDS-PAGE apparatus bath | ||
#fill the space between the gels with running buffer | #fill the space between the gels with running buffer | ||
#Take off the | #Take off the combs of the gel (take it out gently) | ||
#Load the sample to each well | #Load the sample to each well | ||
#Do not use the wells at the left and right end of the gel | #Do not use the wells at the left and right end of the gel | ||
| Line 26: | Line 29: | ||
##The current the the power decreases in time but not reach zero (if it reaches zero see the troubleshoot) | ##The current the the power decreases in time but not reach zero (if it reaches zero see the troubleshoot) | ||
##For the state type of gel it takes 35 - 40 min | ##For the state type of gel it takes 35 - 40 min | ||
===Troubleshoot=== | |||
#The power drops to zero | |||
* really old buffer | |||
* the space in between the gels is not filled with buffer to the top | |||
Latest revision as of 20:22, 21 July 2022
Running SDS-PAGE
- Make ready the samples to run
- Make ready the sample running buffer
- Make ready the gel
- Have the SDS-PAGE apparatus
- Place the the gel in the SDS-PAGE stand
- Place the stand with the gel in the SDS-PAGE apparatus bath
- fill the space between the gels with running buffer
- Take off the combs of the gel (take it out gently)
- Load the sample to each well
- Do not use the wells at the left and right end of the gel
- fill the SDS-PAGE apparatus bath with running buffer to the bottom of the gel
- Place the cover of the SDS-PAGE
- N.B Make sure the correct plug is connected to the correct electrode (red goes to red and black goes to black)
- Plug the cables to the SDS-PAGE apparatus power source
- Turn of the SDS-PAGE power source
- Set to constant voltage
- Use 200 V
- Press the Run button to start the electrophoresis
- The voltage increases to 200 V from 0 V
- For 4% staking gel and 12.5 % separating gel the current reaches 50-60 mA and the power 10 -12 W
- The current the the power decreases in time but not reach zero (if it reaches zero see the troubleshoot)
- For the state type of gel it takes 35 - 40 min
Troubleshoot
- The power drops to zero
- really old buffer
- the space in between the gels is not filled with buffer to the top