BME 205: Lab 4 Final Review

What does protein production mean in terms of this lab?

Popular means of producing recombinant proteins (proteins not naturally found in that organism)

What are some advantages of using E. Coli for protein production?

1. Fast growth kinetics2. Easily reach high cell density culture3. Transformation with exogenous DNA is fast and easy

What is the T7 promoter?

Sequence of DNA that indicates the start of transcription

What is the intermediate between DNA and protein?

RNA

How does DNA turn into RNA?

Transcription

How does RNA turn into a protein?

Translation

What are the essential steps of translation?

mRNA is read and a tRNA attaches the amino acid sequences that correspond to each codon. The amino acids are connected by a polypeptide chain

What are the essential steps of transcription?

Polymerase melts the H bonds between the double-stranded DNA. Polymerase moves in the 3' to 5' direction on the DNA so it can be synthesized in the 5' to 3' direction.

How many mRNA are made from a double-stranded DNA?

One

What does cell lysis mean?

The breaking down of the membrane of a cell

What is a lysate?

Fluid containing the cell components

What is detergent lysis?

Detergent is used to create pores in the membrane by solubilizing membrane lipids and proteins

What is enzymatic lysis?

Enzymatically disrupting cell membrane

What is mechanical lysis?

Using physical force to break down a cell membrane

How did we break apart the cell membrane?

Mechanical lysis; shear force; bead-beating method in the OmniLyse cartridge connected to a battery (syringe up and down)

What is protein purification?

Chromatography separates the target protein from other molecules or compounds in the cellular mixture

What components of the solution elute from the column in chromatography?

Components that do not interact/interact the least amount elute through the column much quicker than the components that interact with the column components.

What are some examples of chromatography types?

Gel filtration, ion exchange, hydrophobic interaction

What does IMAC stand for?

Immobilized Metal Affinity Chromatography

What is the IMAC based on?

IMAC is based on the ability of some amino acids (histidine) to coordinate bonds to metals

Do naturally occurring sequences have the ability to coordinate bonds with metal ions?

No, which is why we must used the modified emGFP gene

What makes emGFP special in regards to IMAC?

emGFP has a peptide of 6 repeating histidines (His-Tag) that will bind to the stationary phase at a pH of 7.5 - 8

How do we remove the emGFP protein from the stationary phase?

We will lower the pH of the column to between 4 and 5 which strips the metal from the polymer resin

What wavelength does the spectrophotometer read at?

280 nm

Why do we use the spectrophotometer to measure?

Fast and convenient (no additional reagents are necessary)

What must we assume about the relationship between [protein] and absorbance?

There is a linear relation but we must account for the extinction coefficient

What does ELISA stand for?

Enzyme Linked Immunosorbent Assay

What does ELISA do

What is a clinical application of ELISA?

Determine if a patient has HIV

What does BCA stand for?

Bichoninic Acid Assay

What is a BCA assay used for?

Quantify the amount of total protein in a sample

What are the two critical reactions in a BCA assay?

1. Peptide bonds in protein reduce Cu2+ ions to Cu+ 2. Two molecules of BCA chelate with each Cu+ ion, which forms a purple colored complex that strongly absorbs light

What is the wavelength that BCA assay absorbs light at?

562 nm

What are some sources of error in a BCA assay?

1. Incompatible contaminants2. Reagents that chelate metal ions, change pH, or reduce copper3. Incompatible [substance]

What does the spectrophotometer tell us about the BCA assay?

Measures the absorbance of the purple-colored complex from the reaction between BCA and Cu+ ions

What is absorbance proportional to in the spectrophotometer reading?

Protein amount

What does ELISA do?

Used to verify the presence of a specific antibody or antigen in a solution, often at low concentrations

What does SDS PAGE stand for?

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

What is the type of gel used in SDS PAGE?

Polyacrylamide Gel

What does SDS PAGE do?

Separates proteins based on size, but using a tighter matrix than agarose gel electrophoresis

What is the importance of SDS in SDS PAGE?

Denatures the protein and lines up the negative charge along the protein polypeptide chain

What is the importance of the reducing agent in SDS PAGE?

Breaks up the cysteine disulfide bonds to aid in protein unfolding

What are the steps of SDS PAGE?

1. Set up electrophoresis unit2. Load boiled samples into their wells of the polyacrylamide gel3. 40 V for 10 minutes4. 200 V for 10-15 minutes5. Shake for 30 minutes on the plate shaker after adding GelCode Blue Safe Protein Stain6. Image on the Azure C400 Plate Reader

How do you prepare a sample for SDS PAGE and why?

Heat the protein for 5 minutes at 95 C to denature the protein