MBG 3350 Midterm 2 Exam Questions with Correct Answers Latest Update 2025/2026
Kaelin, Ratcliffe, Semenza - Answers Nobel Prize (2019) for how cells sense oxygen
James Peebles - Answers Canadian that won Nobel Prize in Physics (2019)
Urich Karl Laemmli - Answers SDS-PAGE
Temin and Baltimore - Answers Reverse Transcriptase (1975)
*discovered independently*
gel filtration chromatography - Answers separates proteins based on size
Applications of Gel filtration chromatography x3 - Answers 1. High capacity but limited in MW
selectivity
2. Can be used to separate large proportion contaminates of different size
3. Can use to quickly desalt - using a column with small exclusion limit
Protein Tags - Major categories - Answers 1. Glutathione Transferase
2. Metal Tagging (IMAC)
3. Antibody - Antigen
Co-immunoprecipitation - Answers A technique to determine whether two or more proteins are
associated with each other can be in column or batchwise
Affinity Chromatography - Answers One step, highly specific interactions
Protein elution through contrasting parameters or competitive inhibitors
affinity chromatography - example Glutathione Transferase - Answers 1. GST tagged with
protein
2. GST with tag binds to the thione on column
3. Wash contaminates
4. Introduce thione for competitive inhibition
5. Use dialysis to separate thione from tagged protein
affinity chromatography - example His-Tag Nickle Column - Answers 1. Poly-His protein binds to
Ni on Ni-NTA column
2. Wash out contaminats with increasing concentration of imidazol
, 3. Use high concentration of competitive inhibitor imidazol to elute protein of interest
4. Regenerate column
5. Can remove his tag after
Affinity Chromatography - Example Antibody - Answers
Protein Quantification methods x3 - Answers 1) A 280
2) BCA assay
3) Coomassie/Bradford Assay
A280 - Answers - aromatic side chains exhibit strong UV absorption
-prone to error and interference with substances in solution
BSA - Answers bovine serum albumin - universal reference
BCA Assay - Answers - Bicinchonic acid
-Protein copper chelation + secondary detection of reduced copper
-Compatable with samples contaning less then 5% surfactants
-Disadvantage: many compounds and reducing agents can interfere
BGG - Answers Bovine Gamma Globin - accepted reference protein for total protein
quantification of purified antibodies or immunoglobin rich samples
Coomassie / Bradford Assay - Answers -protein must be soluble at acidic pH
-colour change due to dye binding is detected at 595nm
-Binding to positively charged aa
Advantages: Fastest and easiest way
- compatible with most salts, buffers, reducing substances, surfactants
? used to solubilize membrane protein with Pierce Assay
Reasons that protein quantification is useful - Answers - allows for equal loading on protein
amounts
When is loading equal volumes useful (example) - Answers - comparing wash steps
SDS-PAGE (definition) - Answers sodium dodecyl sulfate polyacrylamide gel electrophoresis
Kaelin, Ratcliffe, Semenza - Answers Nobel Prize (2019) for how cells sense oxygen
James Peebles - Answers Canadian that won Nobel Prize in Physics (2019)
Urich Karl Laemmli - Answers SDS-PAGE
Temin and Baltimore - Answers Reverse Transcriptase (1975)
*discovered independently*
gel filtration chromatography - Answers separates proteins based on size
Applications of Gel filtration chromatography x3 - Answers 1. High capacity but limited in MW
selectivity
2. Can be used to separate large proportion contaminates of different size
3. Can use to quickly desalt - using a column with small exclusion limit
Protein Tags - Major categories - Answers 1. Glutathione Transferase
2. Metal Tagging (IMAC)
3. Antibody - Antigen
Co-immunoprecipitation - Answers A technique to determine whether two or more proteins are
associated with each other can be in column or batchwise
Affinity Chromatography - Answers One step, highly specific interactions
Protein elution through contrasting parameters or competitive inhibitors
affinity chromatography - example Glutathione Transferase - Answers 1. GST tagged with
protein
2. GST with tag binds to the thione on column
3. Wash contaminates
4. Introduce thione for competitive inhibition
5. Use dialysis to separate thione from tagged protein
affinity chromatography - example His-Tag Nickle Column - Answers 1. Poly-His protein binds to
Ni on Ni-NTA column
2. Wash out contaminats with increasing concentration of imidazol
, 3. Use high concentration of competitive inhibitor imidazol to elute protein of interest
4. Regenerate column
5. Can remove his tag after
Affinity Chromatography - Example Antibody - Answers
Protein Quantification methods x3 - Answers 1) A 280
2) BCA assay
3) Coomassie/Bradford Assay
A280 - Answers - aromatic side chains exhibit strong UV absorption
-prone to error and interference with substances in solution
BSA - Answers bovine serum albumin - universal reference
BCA Assay - Answers - Bicinchonic acid
-Protein copper chelation + secondary detection of reduced copper
-Compatable with samples contaning less then 5% surfactants
-Disadvantage: many compounds and reducing agents can interfere
BGG - Answers Bovine Gamma Globin - accepted reference protein for total protein
quantification of purified antibodies or immunoglobin rich samples
Coomassie / Bradford Assay - Answers -protein must be soluble at acidic pH
-colour change due to dye binding is detected at 595nm
-Binding to positively charged aa
Advantages: Fastest and easiest way
- compatible with most salts, buffers, reducing substances, surfactants
? used to solubilize membrane protein with Pierce Assay
Reasons that protein quantification is useful - Answers - allows for equal loading on protein
amounts
When is loading equal volumes useful (example) - Answers - comparing wash steps
SDS-PAGE (definition) - Answers sodium dodecyl sulfate polyacrylamide gel electrophoresis
