Electrophoresis

Question 1

What is the isoelectric point?

Answer 1

Where the protein has no net charge at that pH

Question 2

What is the charge if pH

Answer 2

Net positive charge

Question 3

What is the charge if pH>pI?

Answer 3

Net negative charge

Question 4

What is pI?

Answer 4

Isoelectric point

Question 5

What are the principles of electrophoresis?

Answer 5

Migration of charged particles in the electric field

Question 6

What is electrophoresis useful for?

Answer 6

Separating/ Purifying macromolecules

Question 7

What does migration of charges depend on in electrophoresis?

Answer 7

Charge, size or shape

Question 8

What gel does the horizontal electrophoresis apparatus normally use?

Answer 8

Agarose

Question 9

What gel does the vertical electrophoresis apparatus normally use?

Answer 9

Polyacrylamide

Question 10

What is the function of the tank in electrophoresis?

Answer 10

Where the sample and buffer are attached to a power block

Question 11

What is the function of the power block in electrophoresis?

Answer 11

Supplies electric current through the buffer

Question 12

What is the function of the casting tray in electrophoresis?

Answer 12

Preparing the gel

Question 13

What is the function of the sample comb in electrophoresis?

Answer 13

Makes an indentation in the gel that allows you to put your sample in the buffer before applying the current

Question 14

What do you need to check when choosing apparatus (3)?

Answer 14

Uniform electric field across gel
Cooling to present thermal artefact
Access to gel loading an monitoring

Question 15

Where is gel electrophoresis usually cast?

Answer 15

In a thin slab within wells

Question 16

What does the buffer provide in gel electrophoresis (3)?

Answer 16

• ions to carry current
• relatively constant pH
• pH of solution and nature of R-groups have an important effect on migration of proteins

Question 17

What is agarose made up of?

Answer 17

Polysaccharide extract from seaweed

Question 18

How is agarose prepared?

Answer 18

Dissolving powdered agarose into buffer. Heat in microwave and pour into a casting tray.
Undergoes polymerisation when cooled

Question 19

Why does agarose have a low resolving power?

Answer 19

Big pores

Question 20

What is agarose used to separate (specifically what size)?

Answer 20

Large proteins >200kDa

Question 21

What concentration is agarose used at?

Answer 21

0.5-2%

Question 22

What is polyacrylamide formed from?

Answer 22

The small synthetic molecule acrylamide

Question 23

What does acrylamide need to be in the presence of to polymerise?

Answer 23

Catalyst and initiator (APS and TEMED)

Question 24

What is the pore size of acrylamide determined by?

Answer 24

Polyacrylamide concentration

Question 25

What does TEMED stand for?

Answer 25

Tetra methyl ethylene diamine

Question 26

What is the most popular technique for protein electrophoresis?

Answer 26

Vertical slab gel

Question 27

What do buffers do in electrophoresis (3)?

Answer 27

Supply current carrying ions Maintains desired pH Provides medium for heat dissipation

Question 28

What are the two classifications for buffers?

Answer 28

Continuous | Discontinuous

Question 29

What is a continuous buffer?

Answer 29

Uses same buffer in gel, sample and tank

Question 30

What is a discontinuous buffer (4)?

Answer 30

Different buffer Non-restrictive large-pore gel Resolving gel -greater restriction Have different buffers for stacking gel, resolving gel and tank buffer

Question 31

What does protein electrophoresis depend on?

Answer 31

The migration of any protein in electricity field depends on pI and pH

Question 32

What happens to the pI of any given protein?

Answer 32

Constant

Question 33

What does the pH of a solution determine in protein electrophoresis?

Answer 33

The charge expressed by the protein

Question 34

What does SDS-PAGE stand for?

Answer 34

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Question 35

What type of detergent does SDS-PAGE have?

Answer 35

Strong anionic

Question 36

Why is the strong anionic detergent important in SDS-PAGE?

Answer 36

- to solubilise, dissociate and denature most proteins to single polypeptide chains

Question 37

What does SDS-PAGE do to hydrogen bonds?

Answer 37

Disrupts them

Question 38

What does SDS-PAGE do to hydrophobic interactions?

Answer 38

Blocks them

Question 39

To what ratio of SDS: protein does SDS-PAGE bind?

Answer 39

1.4:1

Question 40

What cleaving agents does SDS-PAGE include (with example)?

Answer 40

Disulfide bond (beta-mercapto ethanol)

Question 41

What is the migration of the protein determined by in SDS-PAGE?

Answer 41

Weight

Question 42

Where do negatively charged molecules migrate towards in SDS-PAGE?

Answer 42

Positive pole / anode

Question 43

What determines the effective separation range of SDS-PAGE?

Answer 43

Conc of gel

Question 44

What is SDS-PAGE not suitable for?

Answer 44

Small polypeptides and peptides with a molecular weight of less than 10kDa

Question 45

What type of buffer system can be used in protein gel electrophoresis?

Answer 45

Either (continuous or discontinuous)

Question 46

What are the three protein stains you can use?

Answer 46

Coomassie brilliant blue Fluorescent stain silver stain

Question 47

How does the fluorescent stain work?

Answer 47

Use a fluorescent light to see the samples

Question 48

What are the ratios of coomassie brilliant Blue needed in electrophoresis?

Answer 48

9:9:2 v/v/v | Methanol, distilled water and acetic acid

Question 49

How do you compare your sample to known proteins in protein electrophoresis?

Answer 49

You have a band of known proteins running down the side

Question 50

When is native gel electrophoresis used?

Answer 50

Mainly in circumstances where native conformations are to be analysed (as you don’t have to denature the proteins for it to work)

Question 51

What is the separation based on in native gel electrophoresis (2)?

Answer 51

Charge: size ratio and conformation (shape)

Question 52

Give the advantages of native gel electrophoresis (3)

Answer 52

- Potential of separating proteins of identical molecular weight not resolved with SDS-PAGE - recovery of protein in native state - study binding events (protein- protein or protein- ligand)

Question 53

What are the types of nature gels?

Answer 53

Agarose and polyacrylamide

Question 54

What does SPEP stand for?

Answer 54

Serum protein electrophoresis

Question 55

What does SPEP measure?

Answer 55

Specific proteins in blood

Question 56

How does SPEP work?

Answer 56

Uses an electrical field to separate proteins into groups of a similar size, shape and charge

Question 57

What are the two main groups in blood serum?

Answer 57

Albumin | Globulins

Question 58

What is a densitometer?

Answer 58

Used to scan separated proteins in the gel- the patterns give info about protein fractions

Question 59

What is the pH range of haemoglobin electrophoresis?

Answer 59

8-9 (so slightly alkaline)