User:Floriane Briere/Notebook/CHEM-496/2011/10/25: Difference between revisions
From OpenWetWare
m (→Protocol) |
mNo edit summary |
||
(5 intermediate revisions by the same user not shown) | |||
Line 14: | Line 14: | ||
To do so, we are going to run a SDS-PAGE using a 12% discontinuous polyacrylamide gel. | To do so, we are going to run a SDS-PAGE using a 12% discontinuous polyacrylamide gel. | ||
# The SDS-PAGE technique permits to separate proteins according to their molecular weight. The SDS (Sodium Dodecyl Sulfate) is an anionique surfactant which is going to denature the proteins and add negative charges on it; so proteins are going to migrate through the gel according to their molecular weight/negative charge ratio. | |||
# The discontinuous polyacrylamide gel is composed of two parts: | |||
## The stacking gel: compare to the resolving gel, the stacking gel has a lower concentration of acrymalide and a lower pH. It allows to concentrate the proteins into a thight band. | |||
## The resolving gel: it permits to separate the proteins which are going to migrate through the gel according to their molecular weight. | |||
The protocol was esthablished using this [http://ergo.berkeley.edu/be115/protean3.pdf manuel]. | |||
==Protocol== | ==Protocol== | ||
Line 35: | Line 39: | ||
## Add the comb | ## Add the comb | ||
## Let the gel polymerize for 20 minutes | ## Let the gel polymerize for 20 minutes | ||
# Prepare the samples: | |||
## Tube 1: 10µl of ladder (blue) + 4µl of loading buffer (orange) | |||
## Tube 2: 16µl of protein 1 + 4µl of loading buffer (orange) | |||
## Tube 3: 16µl of protein 2 + 4µl of loading buffer (orange) | |||
## Tube 4: 16µl of protein 3 + 4µl of loading buffer (orange) | |||
# Run the SDS-PAGE electrophoresis: | |||
## Remove the comb | ## Remove the comb | ||
## Load the sample into the wells | ## Load the sample into the wells | ||
## Run the gel for 35 minutes at 200 volts | ## Run the gel for 35 minutes at 200 volts | ||
# Stain the polyacrylamide gel: | |||
## Cover the gel with stain (blue) | |||
## Leave overnight on the shaker | |||
[[Category:Course]] | [[Category:Course]] |
Revision as of 09:33, 26 October 2011
File:BDLlogo notext ir.png Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
ObjectiveToday's experiment consists on performing a gel electrophoresis on MPB-intein fusion which have been expressed and purified by groups 2 and 3. This experiment will help us to determine whether or not the expressions and purifications worked well. To do so, we are going to run a SDS-PAGE using a 12% discontinuous polyacrylamide gel.
The protocol was esthablished using this manuel. Protocol
|