Lab Final Flashcards
What is Microplusin?
Antimicrobial Peptide
What are antimicrobial peptides?
Peptides that help attack bacteria
Is the antimicrobial peptide that we are working with anionic or cationic?
Catatonic
What organism is Microplusin derived from?
Isolated from the cell-free hemolymph and egg of the tick Boophilus microplus
What is PRSETA?
It is a vector used for high level protein expression
PRESTA has an antibiotic resistance gene to what antibiotic?
Ampicillin
What are competent cells?
Cells that can uptake foreign DNA
Define transformation.
Foreign DNA introduced into a cell
What type of E. coli were used the transformation?
BL21
What media was used to grow the transformed cells?
LB Media
What antibiotic was used?
Ampicillin
Describe the Lac Operon
Cluster of genes used to regulate lactose
What is heat shock and why is it performed?
To form pores in the cell for DNA uptake
What is IPTG? How does it work?
IPTG is an inducer that mimics lactose and is used to induce protein expression
For how many hours did we incubate the induced protein after induction with IPTG?
4 hours
True or False. In order to express a gene that is controlled by the T7 promoter, you need to add IPTG to block the function of the lac repressor.
True
Name the method for colorimetric detection and total quantification of protein.
BCA assay
How does the BCA assay work? What does BCA stand for?
bicinchoninic acid assay; works by measuring the level of amino acid composition of the protein.
BCA is what type of reaction? Explain.
Colometric measure the absorption
True or False. The total quantification of the protein was measured in absorbance
True
What does SDS-PAGE stand for? How does it work?
Polyacrylamide gel electro
What is SDS? What does it do to proteins?
Sodium deodecyl Sulfate gives a negative charge to proteins
What are the metals used for greatest affinity for histidine?
Nickel
How many histidine does PRSETA have?
6
What does IMAC stand for?
Immobilized metal affinity chromatography
How does the nickel resin column work? What does the histidine tag bind to?
His-tag binds tightly to the immobilized metal ions because the side chain of Histidine.
What does imidazole bind to? What does imidazole compete with when loaded into the nickel resin column?
imidazole, has a specific binding affinity to metal ions (in this case, nickel II). As a result, the desired protein is binded tightly to the beads while other proteins flow through the column easily.
What is bromo-phenol blue?
A pH indicator, a color marker to monitor the progress of polyacrylamide gel electrophoresis.
