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Everything You Need to Know about SDS-Page Gels
Transcript of Everything You Need to Know about SDS-Page Gels
2. Assemble the gel assembly, placing the spacers between the alumina plate and the glass plate. Clamp the plates/spacers onto the gel rig and place the unit on the glass plate.
3. Seal the bottom of the gel assembly with melted 1-2% agarose and allow the agarose to cool.
4. In a 50 mL erylenmeyer flask equipped with a side-arm, pipet the bisacrylamide/acrylamide, H2O, separating (resolving) gel buffer, and 10% SDS into the flask. Add a spatula of the ammonium persulfate and swirl to dissolve the salt. Aspirate the solution briefly to remove dissolved oxygen from the solution.
5. Add 10-15 microliters of TEMED to the solution, twirl it quickly, and quickly pipet the solution in the space between the glass and alumina plates until the solution is about ½ inch from the top of the alumina plate. If desired, pipet a small amount of isopropanol to the top of the gel and allow the gel to polymerize.
6. After the gel polymerizes, pour the isopropanol off of the top of the gel and wash the gel top with some water. Use a papertowel to remove as much of the water as possible. Insert the comb into the top of the gel.
7. In a separate 50 mL erylenmeyer flask equipped with a side-arm, pipet the bisacrylamide/acrylamide, H2O, stacking gel buffer, and 10% SDS into the flask. Add a spatula of the ammonium persulfate and swirl to dissolve the salt. Aspirate the solution briefly to remove dissolved oxygen from the solution.
8. Add 10-15 microliters of TEMED to the solution, twirl it quickly, and quickly pipet the solution in the space between the glass and alumina plates around the comb. Allow this solution to polymerize.
9. Snap the gel rig into buffer chamber and pour 1X TAE (or TBE) buffer into the space behind the gel and in the bottom of the buffer chamber. Carefully remove the comb so you do not destroy the walls of the wells.
10. Load your boiled SDS samples and run the gel at 10-12 A.
Gel Pictures SDS-PAGE- Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis First of all, the object of SDS-Page is to separate different proteins on the basis of size. In order to do this, all of the proteins need to be made linear so shape is not confounding variable acting on separation. SDS- sodium dodecyl sulfate- is a detergent that denatures proteins so that they no longer have secondary, tertiary, or quaternary structure. In effect, SDS breaks up hydrophobic portions in a protein and coats the entire surface of the protein with negative charges. Secondly, polyacrylamide is used as the environment in which the proteins are separated. Polyacrylamide gels are formed from the polymerization of two compounds, acrylamide and N,N-methylene- bis-acrylamide (Bis, for short). Bis is a cross-linking agent for the gels. The polymerization is initiated by the addition of ammonium persulfate along with N,N,N,N,- tetramethylethylenediamine (TEMED). The gels are neutral, hydrophillic, three-dimensional networks of long hydrocarbons crosslinked by methylene groups. Since the polyacrylamide creates a maze of tunnels and spaces, smaller proteins move faster through the gel as opposed to bigger proteins. As the total amount of acrylamide increases, the pore size decreases and smaller molecules are more easily separated. The percent gel to use depends on the molecular weight of the protein to be separated. Use 5% gels for proteins ranging from 60,000 to 200,000 daltons, 10% gels for a range of 16,000 to 70,000 daltons and 15% gels for a range of 12,000 to 45,000 daltons. The proteins tend to move the gel in bands, and therefore after a gel is dyed, one can compare the bands to the molecular marker to see if pure protein was obtained. http://www.ruf.rice.edu/~bioslabs/studies/sds-page/sdsgoofs.html Acrylamide is toxic when ingested and can be absorbed through the skin. Once the acrylamide is polymerized, it is no longer absorbable, but care still should be taken when disposing of the gel.SDS-PAGE separates proteins based on their primary structure of size, but not amino acid sequence. Therefore, if we had many copies of two different proteins that were both 500 amino acids long, they would travel together through the gel in a mixed band. As a result, we would not be able to use SDS-PAGE to separate these two proteins from each other. Polyacrylamide gels were first used in the 1960s after scientists proved their versatility over starch gels. Thus, PAGE was born. Shapiro et al. (1967) were the first to make use of SDS in PAGE. Then, in 1970, Laemmli showed that proteins could be fractionated well with SDS-PAGE. Weber and Osborn (1969) demonstrated that proteins moved well with their molecular weights. After the success of SDS-PAGE, people began to use gels in tubes, then as slabs and subsequently as minigels.