Lecture 7 - Cellular and molecular techniques Flashcards Preview

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Flashcards in Lecture 7 - Cellular and molecular techniques Deck (24):
1

What are the three steps in function investigation using mutation?

1. Design primers to introduce a mutation
2. Confirm mutation using sequence
3. Express a protein to check function

2

What can we do with DNA vs RNA?

DNA can be used to transfect cells and RNA can be used to inject oocytes

3

What can we do with the purified protein produced from cells (2)?

Crystallography studies
Liposome reconstitution

4

Why use E.Coli cells for protein expression vs human mammalian cells?

The higher the complexity of the systems, the longer it takes to double/reproduce the cell

5

How to arabinose and IPTG induce over-expressioin

They bind to the inhibitor of the gene to allow the polymerase to bind to the DNA to translate it

6

How are his-tags used to purify proteins?

1. Cells are crushed, concentrated, spun and run through a column
2. Proteins with his-tag will attach to the column
3. Column is washed with buffer
4. Protein is eluted using a molecule with a higher affinity to NTA/nickel (imidazole)

7

What are the four characteristics of a protein for it to crystallise?

Pure/homogenous
Stable
Soluble
Voluminous

8

How is the his-tag removed

Thrombin digest

9

What are the two crystallography studies that can be done with purified protein?

Binding assay
Isothermal titration calorimetry

10

What does isothermal titration calorimetry measure?

Thermodynamic parameters in a solution between a ligand and a protein

11

What type of assay is used to examine the binding of a ligand or substrate?

Fluorescence based assay, intrinsic F or introduced F

12

What can be measured in a liposome reconstitution assay with GLTph?

The uptake of glutamate and the movement of sodium which is required for the uptake.

13

What is protein crystallography?

The visualisation of protein structures at atomic state resolution

14

What does protein crystallography allow us to do? (2)

Understand conformational changes in proteins
Develop drugs to specifically bind to proteins

15

What EMR does protein crystallography use?

X-rays

16

Why do we require crystals for amplification of signal in x-ray crystallography?

Crystals have many unit cells and there is more scattering

17

How does a synchrotron produce EMR

By accelerating electrons almost to the speed of light

18

Are hydrophobic proteins membrane bound or soluble generally?

Membrane bound

19

Why are membrane proteins difficult to perform x-ray crystallography for?

They are difficult to crystallise because of the difficulty of removing them from the membrane

20

What is required to solubilise the membrane proteins?

Detergent

21

What is the human glutamate transporter

An excitatory amino acid transporter

22

What information do we have on the human glutamate transporter (4)?

DNA sequence
Amino acid-sequence
Hydropathy plot
Biochemical techniques - 2D topology

23

What is used to determine function and structure of EAATs instead?

GltPh

24

What information have we gained from crystallography of the homologue?

Transmembrane domains/topology
Residues implicated in substrate and ion-binding (in C-terminus)