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1

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)

2

SDS-PAGE is an analytical tool for what?

1) Determining the purity of protein preparations
2) Approximate molecular weight determination
3) Western Blot analysis

3

Migration of proteins in SDS-PAGE is relatively proportional to?

The mass of the proteins.

4

Smaller molecules travel ______ than larger molecules in SDS-PAGE.

farther

5

How are protein bands visualized in SDS-PAGE?

Staining by Coomassie Brilliant Blue/Silver Staining or by immunoblotting.

6

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.

7

What is the Rf value?

Ratio of the distance migrated by the protein over the distance traveled by the dye font.

8

The SDS-PAGE lab objective was to analyze the success of what?

1) IPTG induction
2) Cell lysis efficiency
3) Solubility analysis

9

What is 2D gel electrophoresis?

Combines both IEF and SDS-PAGE. Provides a fingerprint of protein expression (Crude) in a given cell.

10

2D-Gel electrophoresis is a core technique in what?

Systems Biology

11

What are the shortcomings of 2D-gel electrophoresis?

Inherent gel to gel variability. (Non-biological variation)

12

2D-gel electrophoresis is an example of _________ separation.

Orthogonal

13

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.

14

What is a protein's pI?

Isoelectric point. pH at which a protein has a neutral charge.

15

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.

16

DIGE is often used for whatt?

Often used to see changes in protein abundance between healthy and diseased individuals, changes in PTM, truncations and other modifications that could alter the size or pI of proteins.

17

DIGE has only a narrow, binary range of possible changes. What are they?

Vertical: change in size or molecular weight.
Horizontal: change in pI
Diagonal: Change in both.

18

Reciprocal labeling is done for what reason? (DIGE)

To make sure the changes seen between samples are not due to dye-dependent interactions.

19

What is image analysis in DIGE?

Superimposed picture analysis by a computer overlay.

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