Week 2 - Protein Chromatography Flashcards

Question

PROTEIN PURIFICATION - METHOD TO CHECK THE PRODUCT PURITY

Answer 1

find out the isoelectric point (pI) Approx mw

Answer 2

Electrophoresis –Polyacrylamide gel – a molecular “sieve” – slowing migration of proteins in proportion to charge to mass ratio as below

Answer 3

- Common method for estimating purity and mw - Uses SDS - Binding ratio 1.4x – nearly 1 molecule of SDS to each aa residue - Sulfate moiety of bound SDS contributes to net negative charge – intrinsic charge of protein not significant - SDS binding partially unfolds proteins – rodlike shapes - Separation mainly on basis of mw - Visualisation – Coomassie blue binds to proteins

Answer 4

- Chromatofocusing medium equilibrated with a start buffer at a pH slightly above the highest pH required. - Sample is applied to the chromatographic column with the start buffer - Elution buffer is passed through the column and begins to titrate the amines on the medium and the proteins – gradient pH develops - Proteins in the sample that are at a pH above their pI are negatively charged and retained near the top of the column - The ones having their pH below pI begin to migrate down and bind to that part of the column where the pH is above their pI

Answer 5

- Binding dependent on the surface charge of the protein - Uses ion exchange resins - Uses FPLC - Elutes bound species by altering the pH of the buffer - Proteins elute in order of their isoelectric points.

Answer 6

- More sensitive than either alone.. - separates proteins of identical molecular weight that differ in pI, orproteins with similar pI values but different mw

Answer 7

- Lyophilization aka. freeze drying - Ultrafiltration - Chromatographic concentration - Precipitation

Answer 8

- Enzyme assay - Protein assay - SDS-PAGE - Western Blotting

Answer 9

SDS-PAGE & immunoblotting

Answer 10

- Manipulated form of native protein - Generated in various ways in order to increase production of proteins, modify - Coding sequence for the protein of interest is isolated and cloned into an expression plasmid vector. - Most recombinant proteins for therapeutic use are from humans but are expressed in microorganisms such as bacteria, yeast, or animal cells in culture. - intron-free version of the gene is often made by converting the mRNA into cDNA - But cDNA lacks regulatory regions so expression vectors provide promoter, ribosome-binding site, and terminator sequences

Answer 11

Lab techniques – - ELISA – matched antibody pairs – standards - Western Blot – positive controls - Immunohistochemistry - Enzyme assays - Cellular responses to stress and disease - In animal models – can help with identifying therapeutic targets

Answer 12

First use – insulin (1982) Now –recombinant hormones, interferons, interleukins, growth factors, tumor necrosis factors, blood clotting factors, thrombolytic drugs,treating major diseases Enzymes – animal feed enhancement Lactic acid bacteria – used for fermenting foods, now adapted for use in human/animal digestion and nutrition

Answer 13

Ethical considerations Quick Cost Scaling

Answer 14

Contamination, e.g. proteasesInactive protein, e.g. inclusion bodies Small proteins only Lack of post-translational modifications

Answer 15

catch different parts of solution or gas we are measuring eg. paper where we pass liquid over it

Answer 16

load sample with solution let it run through

Answer 17

the thing that is absorbing packing material

Answer 18

we need to equilibrate column this is done by putting solvent through column eg. water or petrol so column is bathed in whatever the solute is going to dissolve in

Answer 19

polar compounds

Answer 20

uses osmotic effects to draw out small solutes

Answer 21

resin - charged

Answer 22

proteins move through depending on charge

Answer 23

proteins with negative charge move faster ad come out earlier

Answer 24

separates protein molecules by size - larger molecules come out earlier as smaller molecules are trapped in the beads

Answer 25

by forming ligands - they trap molecules of interest as it goes down the column

Answer 26

change binding conditions proteins separated out differently 2 peaks formed

Answer 27

for ligand attachment matrix should be chemically and physically inert

Answer 28

used to improve binding between ligand and target molecules by overcoming any effects of steric hindrance

Answer 29

molecule that binds reversibly to a specific target molecule or group of target molecule

Answer 30

affinity medium is equilibrated in binding buffer

Answer 31

sample is applied under conditions that favour specific binding of the target molecules(s) to a complementary binding substrate (the ligand) target substance bind specifically but reversibly to the ligand and unbound material washes through the column

Answer 32

target protein is recovered by changing conditions to favour elution of bound molecules elution is performed specifically using a competitive ligand or non-specifically by changing the pH, ionic strength or polarity target protein is collected and purified, concentrated form

Answer 33

affinity medium is re-equilbrated binding buffer

Answer 34

equilibrium adsorption of sample and elution of unbound material wash away unbound material elute bound protein(s) re-equilibration

Answer 35

salt concentration is lowered gradually and samples will elute from column in order of hydrophobicity

Answer 36

absorbance

Answer 37

column volumes (CV)

Answer 38

as we move along, more tightly bound molecules therefore we change salts

Answer 39

helps precipitate out any addition of proteins

Answer 40

better binding

Answer 41

bind gene encoding on target protein so we recognise it at the end tag can be put at either ends

Answer 42

we use its properties to separate our target

Answer 43

acts as molecular signal to filter different proteins

Answer 44

type of detergent

Answer 45

type of polyacrylamide gel

Answer 46

run vertically rather than horizontally

Answer 47

very good for binding to protein tells us where proteins are coming out of gel

Answer 48

bind proteins of interest with primary antibodies and a conjugate with a dye

Answer 49

take sequence and amplified part of reproductive processes

Answer 50

from humans

Answer 51

we need promotor regions

Answer 52

cDNA does not have regulatory genes

Answer 53

Beads in the column - covalently attached ligand

Answer 54

Any protein with affinity for the ligand binds to the beads - migration retarded Proteins that do not bind flow more rapidly through the column Bound proteins eluted by a solution containing either a high concentration of Free ligand competes with the ligand attached to the beads, releasing the protein product that elutes from the column is often bound to the ligand used

Week 2 - Protein Chromatography Flashcards

(79 cards)