Although the technique is not particularly suited for isolating or purifying proteins, it is extremely useful for- i). This technique separates charged molecules in an electric field. Electrophoresis: General Principles Electrophoresis is an analytical technique commonly used in biochemical laboratories. pipette small volumes confidently INTRODUCTION A. comment on the sensitivity of Coomassie blue and predict optimal protein amounts You should also be able to 1. compare experimental molecular weight estimates to literature values 11. estimate the molecular weights of the polypeptides from a standard curve 10. stain (and destain) gels with Coomassie blue 8. be confident in preparing samples for SDS-PAGE and loading samples onto gels and be able run a gel and know when to stop the run 7. understand the function of SDS, heating, and b–mercaptoethanol have on protein structure and be able to predict polypeptide mobility on an acrylamide gel if these were omitted. appreciate the relation between amount of sample loaded and band intensity 5. deduce/predict protein quantarternary structure based on protein MW and polypeptide bands observed on gel d). estimate the molecular weight of the polypeptide chains c). Insert a comb into the unpolymerized stacking gel and allow it to set. For the sake of simplicity, fill the glass sandwich plates all the way to the top. estimate the number of polypeptides chains in a sample b). Add the 10 APS and TEMED to the stacking gel ingredients, mix, and pour immediately on top of the resolving gel. explain how a stacking gel can impact resolution 4. appreciate how sample amount contributes to resolution b). understand the term resolution in the context of a gel electrophoresis a). predict what percentage of polyacrylamide gels should be used for large molecular weight polypeptides versus small molecular weight polypeptides. appreciate variables which influence the mobility of polypeptide chains in an acrylamide matrix. At the end of this experiment you should be able to- 1. Into Intro to SDS-PAGE/Biochemistry I Lab 30 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis _ OBJECTIVES: To estimate the molecular weight of proteins separated on a SDS-polyacrylamide gel.
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