BMI_Lab11_ProteinAnalysis

BMI_Lab11_ProteinAnalysis - Laboratory XI Topic Protein...

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Laboratory: # XI Topic: Protein Determination and Electrophoresis Introduction: Coomassie Blue dye complexes with proteins proteins to give blue color. The Coomassie Blue dye –protein complex is used to assay proteins in solution and visualize proteins separated on polyacrylamide gels. Several modifications of the Coomassie Blue dye reagents are available that enhance color of the reaction and to increase sensitivity of the assays (Lowry, Bardford) and for detection of protein separations (GelCode blue). (The following is excerpted from Instructions for the Bradford Protein Assay) “The Bradford Reagent for protein determination can be used to determine the concentration of proteins. The protein-dye complex causes a shift in the dye absorption maximum from 465 to 595nm. The amount of absorption produced is proportional to the protein concentration. The analysis gives linear response from 1 μ g to 140 μ g based on Bovine Serum Albumin (BSA)”. Serum is a complex mixture of several biomolecules proteins, lipids and minerals) A variety of sera is used in culturing cells. Bovine serum albumin (BSA) is a major component of serum. Today we will use the Bradford to determine the concentration of total protein present in a sample of fetal bovine serum and horse serum. We will also separate the complex mixture of proteins in fetal bovine serum (from cow fetus) and horse serum on a 10% SDS page polyacrylamide gel. The protein molecular marker contains a mixture of six proteins of known molecular weight. The marker is used to determine the size of unknown proteins. Protein Approx. mol. weight Carbonic anhydrase 29,000 Albumin, egg (ovalbumin) 45,000 Albumin, bovine plasma 66,000 Phosphorylase B, rabbit muscle 97,400 subunit B-Galactosidase, escherichia coli 116,000 subunit Myosin, rabbit muscle (0.5mg/vial) 205,000 subunit MATERIALS Before each lab, graduate assistants will prepare protein dilution: 1. Take 3ml of stock BSA (2mg/ml) and q.s. up to 240ml with PBS
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BMI_Lab11_ProteinAnalysis - Laboratory XI Topic Protein...

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