Lecture 7 - Cellular and molecular techniques Flashcards Preview

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

What are the three steps in function investigation using mutation?

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

What can we do with DNA vs RNA?

A

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

3
Q

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

A

Crystallography studies

Liposome reconstitution

4
Q

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

A

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

5
Q

How to arabinose and IPTG induce over-expressioin

A

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

6
Q

How are his-tags used to purify proteins?

A
  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
Q

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

A

Pure/homogenous
Stable
Soluble
Voluminous

8
Q

How is the his-tag removed

A

Thrombin digest

9
Q

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

A

Binding assay

Isothermal titration calorimetry

10
Q

What does isothermal titration calorimetry measure?

A

Thermodynamic parameters in a solution between a ligand and a protein

11
Q

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

A

Fluorescence based assay, intrinsic F or introduced F

12
Q

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

A

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

13
Q

What is protein crystallography?

A

The visualisation of protein structures at atomic state resolution

14
Q

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

A

Understand conformational changes in proteins

Develop drugs to specifically bind to proteins

15
Q

What EMR does protein crystallography use?

A

X-rays

16
Q

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

A

Crystals have many unit cells and there is more scattering

17
Q

How does a synchrotron produce EMR

A

By accelerating electrons almost to the speed of light

18
Q

Are hydrophobic proteins membrane bound or soluble generally?

A

Membrane bound

19
Q

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

A

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

20
Q

What is required to solubilise the membrane proteins?

A

Detergent

21
Q

What is the human glutamate transporter

A

An excitatory amino acid transporter

22
Q

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

A

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

23
Q

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

A

GltPh

24
Q

What information have we gained from crystallography of the homologue?

A

Transmembrane domains/topology

Residues implicated in substrate and ion-binding (in C-terminus)