Lab 1 - Protein Determination

Lab 1 - Protein Determination - Protein Determination...

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Protein Determination INTRODUCTION: Spectroscopy allows for the researcher to view a protein based on how the protein interacts with a reagent. We will be determining what an unknown protein mixed with BSA or Lysozyme based on results found from standards. In order to de4termine the protein we will be using the Biuret, Lowry, and Bradford methods. From these methods we will find that the Biuret and Brandford Assays are faster, the Biuret Assay lacks sensitivity and the Brandford assay can only be used in microscale. Of these methods, the Brandford assay will most likely show the best results in comparison to the other two assays. EXPERIMENTAL: Biuret Solutions: H2O BSA (10 mg/ml) BSA Unknown Reagent Lowry Solutions: H2O BSA (1mg/ml) BSA Unknown Reagent Lysozyme (1mg/ml) Lysozyme Unknown 0.3 mL phenol Bradford Solutions: H2O BSA (1mg/ml) BSA Unknown Reagent Lysozyme (1mg/ml) Lysozyme Unknown Spectrophotometer Micropipette cuvet Biuret Protocol: 1. Acquire 6 test tubes and place into test tube rack 2. From the protein determination protocols page (in appendixes) place the appropriate aliquots of solutions into the test tubes 3. Apply paraffin wrap to the open end of the test tube and mix the solution 4. Place blanking solution into a cuvet and set the spectrophotometer to read absorbance 550 nm 5. After zeroing the spectrophotometer clean the cuvet and place 1.0 mL into the cuvet, then place into spectrophotometer (record reading) 6. Remove and clean cuvet, then repeat step 4 and 5 for remaining samples Lowry Protocol: 1. Acquire 6 test tubes and place into test tube rack 2. From the protein determination protocols page (in appendixes) place the appropriate aliquots of solutions into the test tubes (protocol is done with either BSA or Lysozyme) 3. Apply paraffin wrap to the open end of the test tube and mix the solution 4. After mixing, incubate with reagent for 10 minutes 5. After incubation, add 0.3 mL phenol, incubate for 10 to 30 minutes.
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6. After incubation place blanking solution into a cuvet and set the spectrophotometer to read absorbance 500 nm
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This note was uploaded on 01/26/2009 for the course BIOLOGY 344 taught by Professor Cheng during the Spring '05 term at Temple.

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Lab 1 - Protein Determination - Protein Determination...

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