Biotech Final - Lecture 3 Flashcards
What is the separation of proteins by SDS-PAGE based on?
Based on the movement of charged (negative) proteins in an electrical field in concordance with sieving effects. (separated based on molecular weight)
SDS-PAGE is an analytical tool for what?
1) Determining the purity of protein preparations
2) Approximate molecular weight determination
3) Western Blot analysis
Migration of proteins in SDS-PAGE is relatively proportional to?
The mass of the proteins.
Smaller molecules travel ______ than larger molecules in SDS-PAGE.
farther
How are protein bands visualized in SDS-PAGE?
Staining by Coomassie Brilliant Blue/Silver Staining or by immunoblotting.
A standard curve is often used to appropriate the molecular weight of a protein of interest. What is this curve?
Made from the migration of a molecular weight standard. The Y-axis is the log of the molecular weight whilst the X-axis is the relative migration.
What is the Rf value?
Ratio of the distance migrated by the protein over the distance traveled by the dye font.
The SDS-PAGE lab objective was to analyze the success of what?
1) IPTG induction
2) Cell lysis efficiency
3) Solubility analysis
What is 2D gel electrophoresis?
Combines both IEF and SDS-PAGE. Provides a fingerprint of protein expression (Crude) in a given cell.
2D-Gel electrophoresis is a core technique in what?
Systems Biology
What are the shortcomings of 2D-gel electrophoresis?
Inherent gel to gel variability. (Non-biological variation)
2D-gel electrophoresis is an example of _________ separation.
Orthogonal
What is IEF?
Isoelectric focusing. If a mixture of proteins is electrophoresed through a solution or gel that has a stable pH gradient in which the pH smoothly increases from anode to cathode, each protein will migrate to the position in the pH gradient corresponding to its pI.
What is a protein’s pI?
Isoelectric point. pH at which a protein has a neutral charge.
What is DIGE?
Differential in gel electrophoresis. Two samples of proteins are labeled with different fluorescent dyes (different max absorbance) prior to 2D-gel electrophoresis. The two samples are mixed and loaded onto IEF for first dimension. The strip is then transferred to an SDS-PAGE gel. After SDS-PAGE, the gel is scanned with the excitation wavelength of each dye one after the other, so each sample can be seen separately.
