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Flashcards in Practicals Deck (158)
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1

What is an assay

An indirect method for quantifying a substance or activity of biochemical interest

2

Why is it known as the Bradford Assay

It uses the dye Coomassie Brilliant Blue, which was introduced by Marion Bradford

3

What are the colours associated with Coomassie Brilliant Blue

Green-brown when free in acid solution

Becomes blue when bound to a protein

4

What does LDH do

How can it’s activity be measured

Catalysed conversion of lactate to pyruvate using NAD as a H+ receptor

By following production of NADH

5

Which fixes conducting medium is used for electrophoresis of proteins

How is it prepared

A porous medium of aqueous polyacrylamide

Dissolving acrylamide and a cross linking agent

6

How do you vary the pore size of polyacrylamide gel

Modify the acrylamide monomer concentration during preparation

7

What happens to proteins in the presence of sodium dodecyl sulphate (SDS)

Proteins are completely unfolded

8

Why can inherent differences in charge due to amino acid side chains obscured in presence of SDS

Most proteins bind equal amounts of anionic dodecyl sulphate per gram So behave as polyanions with a constant ratio of negative charge to mass

9

Most proteins bind what amount of anionic dodecyl sulphate per gram

1.4g per gram of protein

10

Why can multimeric proteins be dissociated into their subunits by SDS

Disulphide bonds have been previously reduced by mercaptoethanol

11

During electrophoresis with polyacrylamide gel that contains SDS proteins move at speeds determined by what

The size of their SDS-protein complex

12

What is an isozyme

An enzyme that can exist in more than one molecular form that is usually tissue-specific

AKA isoenzyme

13

How many isoenzymes exist of LDH

Why

Five


because it has four sub units and can be encoded by 2 different genes (A and B) so 5 combinations of A and B are possible

ie. A4, A3B1, A2B2, A1B3, B4

14

How would you find the origin of an LDH isoenzyme in a diseased serum

Compare the activity forms in the gel with that of the LDH from a variety of rat tissue extract by subjecting them to non-denaturing PAGE

Stain the gel with a substrate mixture that turns purple in regions where LDH is found

15

Why is the Bradford assay not appropriate for diagnosis of a mild heart attack

It will not give specific results for the specific isozyme of LDH in the serum

16

What is an antibody comprised of

They are white shaped molecules, comprising four chains, two light and 2 heavy

17

Describe the light and heavy chain components of an antibody

Light: Contains one part of Ig domains

Heavy: Contains two pairs of Ig domains connected by a flexible linker

18

How are the light and heavy chains of an antibody linked together

By a disulphide bond

19

True or false:

Each arm of an antibody will bind to a different antigenic component

False

both find the same antigenic component

20

What produces antibodies

When are the antigen binding sites produced

β cells

During β cell maturation

21

Which three genes are used in a heavy chain locus

What about the light chain

Heavy:
V(variable)
D (diversity)
J (joining)

Light: only V and J

22

What is somatic recombination

Within each cell the V, D, J DNA sequences are randomly recombined during β cell maturation to generate a single V-D-J (or V-J) DNA sequence to form the variable arms of the antibody Y

23

What is an epitope

An antigenic determinant in a protein

24

What happens if a random beta-cell Happens to express an antibody that binds to an antigen of interest

The beta-cell will be stimulated to differentiate and multiply, producing a clone of beta cells, each secreting the same specific antibody against the antigen

25

Can more than one beta-cell be activated by an antigen

Yes

26

The blood from an immunised animal will generally contain a mixture of different antibodies to an antigen. Why is this and what is it called?

It is common for more than one beta-cell to be activated by an antigen

This mixture is known as a polyclonal antibody

27

How do you produce a mono monoclonal antibody

Beta cells must be removed from the spleen of an immunised animal and are fused with cells from a myeloma to produce hybrid cells that grow like cancer cells but produce large amounts of antibody

These antibodies are tested for antigen binding and the cell that produces the tightest binding antibody can then be selected and used to generate a cell line that manufactures large amounts of antibody.

28

Which are more expensive to produce:

Monoclonal antibodies or polyclonal antibodies

Monoclonal

29

True or false:

Monoclonal antibody is can be produced in unlimited quantities

True: parent hybridomas can grow indefinitely

30

What does ELISA stand for

Enzyme linked immunosorbent assay