BiochemII lab report

BiochemII lab report - Wesley Smith Biochem II Lab...

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Wesley Smith Biochem II Lab Report-Spring 2010 Lab 1 (1/25/10): In this lab, the students gained knowledge of protein quantitation, which is basically how to measure protein concentration in a solution. The concentration was determined using the Bradford Assay. The Bradford Assay is a good assay because it relies on Coomassie Brilliant Blue Dye which has dual charge and can be quantitatively absorbed by all proteins. It primarily binds arginines and histidines but also aromatic amino acids best under acidic conditions. A calibration plot was made by making 5 samples each with 1mL of Bradford reagent. Then using a known concentration of protein (1mg/mL) of Bovine Serum Albumin (BSA) 2μL, 5μL, 10μL, 15μL, & 20μL were added to each sample respectively and their absorbance was recorded using a spectrophotometer at 1 min and 2 min intervals. A protein, with unknown concentration, was then given and determined to have an absorbance of 0.152, thus having a concentration of 2.04mg/mL and a molar concentration of 1.8 X 10 -5 M. Lab 2 (2/1/10): In this lab, the students were introduced to SDS/polyacrylomide electrophoresis, which is a key method for the irreversible denaturing separation of proteins. SDS electrophoresis allows one to separate proteins by mass primarily but charge contributes as well. The gel is a mixture of 6-15% polyacrylomide & Bis acrylomide mixture + TEMED (which is responsible for forming cross links between the polymers) + ammonium persulfate (which initiates the cross linking). SDS is a detergent (hydrophobic tail and polar head) that carries a negative charge, so when it binds with proteins (regardless of charge) it makes all proteins negative, though the magnitude of the negative charge may differ. Without SDS or some other agent, negative proteins and positive proteins would separate in different directions on the gel. The gel is divided between a Separating Gel and a Stacking Gel; the primary difference is difference in pH. The separating gel mixture polymerizes as fast as 2-30 minutes, and the stacking gel can polymerize as fast as 1 minute. A protein sample of RNA polymerase was ran for 50 minutes at 30mAmps/gel. The resulting gel was then dyed with colloidal Coomassie Blue dye. Lab 3 (2/8/10): The students were introduced to the valuable procedure of silver staining. Silver staining is a great method for protein detection, as it is very sensitive. It is not however very good for protein quanitification. It is 10-100 fold more sensitive than Coomassie staining. Silver Staining is one of the
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BiochemII lab report - Wesley Smith Biochem II Lab...

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