Shreffler: SDS-PAGE: Difference between revisions
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#Make a 20 µl loading solution for each protein consisting of: | #Make a 20 µl loading solution for each protein consisting of: | ||
*5 µL LDS Sample Buffer (4X) | **5 µL LDS Sample Buffer (4X) | ||
*2 µL Reducing Agent | **2 µL Reducing Agent | ||
*Volume containing 10 µg protein (e.g. if protein stock solution is 1 µg/µl, add 10 µl) | **Volume containing 10 µg protein (e.g. if protein stock solution is 1 µg/µl, add 10 µl) | ||
*Bring up to 20 µl total with PBS. | **Bring up to 20 µl total with PBS. | ||
#Heat samples on heating block (or water bath) 70°C for 10 mins (protein denaturing and SDS binding). | #Heat samples on heating block (or water bath) 70°C for 10 mins (protein denaturing and SDS binding). | ||
Revision as of 11:34, 22 March 2012
Overview
From wikipedia: "SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a technique widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility (a function of the length of a polypeptide chain and its charge). In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis."
We use the Invitrogen NuPAGE system.
Materials
- Appropriate gelbox/voltage source
- NuPAGE 4-12% BIS-TRIS Gel (Invitrogen NP0322BOX)
- NuPAGE MES SDS Running Buffer (20x) (Invitrogen NP0002)
- NuPAGE SimplyBlue SafeStain (Invitrogen LC6060)
- NuPAGE LDS Sample Buffer (4X) (Invitrogen NP0007)
- NuPAGE Antioxidant (Invitrogen NP0005)
- NuPAGE Reducing Agent (Invitrogen NP0004)
Procedure
Preparing Samples
- Make a 20 µl loading solution for each protein consisting of:
- 5 µL LDS Sample Buffer (4X)
- 2 µL Reducing Agent
- Volume containing 10 µg protein (e.g. if protein stock solution is 1 µg/µl, add 10 µl)
- Bring up to 20 µl total with PBS.
- Heat samples on heating block (or water bath) 70°C for 10 mins (protein denaturing and SDS binding).
Running Gel
Discussion
Contact
- Who has experience with this protocol?
- Email Alex Ma through OpenWetWare
