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Bchem 411 > Part II 100-150 > Flashcards

Part II 100-150 Flashcards

1
Q

What is ion exchange chromatography

A

separation based on the reversible interaction between a charged protein and and oppositely charged chromatographic medium

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2
Q

In a cation exhange chromatography what elutes later

A

More postive elites later since more tightly bound

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3
Q

If the matrix is positive charged in ion exchange what type ion exchange is it

A

Anion exhange

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4
Q

In ion exchange what makes the postive charge on the protein

Where do negative charges come from

A

The amino term and the postive charged side chai

The carboxy term and the two negative charged amino acids asp and glu

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5
Q

In an anion exchange what would elite first

Why

A

Less negative charge proteins

This is because the more negative charged need a higher concentration of salt to elute

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6
Q

In ion exchange chromatography what is the column eqwulibrated in

Why

A

A low salt buffer

This lets us load our protein onto the column

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7
Q

What comes out of the ion exhange column right after loading it

A

The unbound molecules (flowthrough)

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8
Q

What is the salt gradient in ion exchange chromatography

A

Low to high

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9
Q

What pH is cation exchange generally run at

Anion

A

Low pH (5.5-6)

Higher pH (8-8.5)

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10
Q

Explain how 3 charged protein in the cation exhange elute different base on ph

A

If the ph is lower, the surface charge on each protein is more postive

The more postive, the tigger they all bind to the matrix and the longer it takes to elite

As ph increase, the surface charges become less positive, the least postive elites earlier since less tightly bound

At some point only one protein carries postive charge and the other two have negative

The negative charges one don’t bind to the column and come out as FT , positive binds but a lot less

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11
Q

In anion exchange, if the charge of the proteins are more similar Wahab does this mean for separation

A

The peaks are less separated since they likely elute at the same time

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12
Q

When ph is above pi of the protein what type of exchanger will that protein bind to

What about below

A

Anion exchanger (since protien is now anionic)

Cation

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13
Q

In ion exchange What does it mean when the ph = pi

A

The net charge of the protein is zero

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14
Q

In ion exhange, as the ph increases where is the first place that the protein lose its charge from

What next

What next

A

The amino terminus (so its deprotonated at the amino terminus)

The side chain of the acidic amino acid (asp or glu)

The side chain of cysteine gets deprotonated

Then the basic amino acids get deprotonated (lys arg his)

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15
Q

What are the strong ion exchangers which are cation and anion

What is the pH range they work at (they are fully charge)

A

Q (anion exchange)

S and SP (cation exchange)

2-12 (broad pH range)

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16
Q

What are the weak ion exchangers which are cation and anion

What is the pH range they work at (they are fully charge)

A

DEAE (anion). : 2-9

CM (cation) : 6-10

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17
Q

What does strong ion exchanger mean

A

If means the column is fully charged over a greater pH range

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18
Q

What does DEAE stand for

A

Diethylaminoethyl

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19
Q

I’m ion exchange chromatography what does the A280 monitor

A
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20
Q

I’m ion exchange chromatography what does the A280 monitor

A

The concentration of protein in the fraction in mg/ml

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21
Q

What happens to the A280 if you have a really put protein

A

The a280 corresponds (lines up) to the activity peak

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22
Q

If there is a diagram on the chromatography profile what do we call it

A

An inset

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23
Q

What is considered a shallow salt gradient in ion exchange chromatography

A

0-200mM

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24
Q

In case study 1 (ion exchange) what we’re they identifying and why

A

Trying to find the kinase (PK) that activate PKB and see if it’s PIP3 dependent

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25
Q

In case study 1 (ion exchange) how did they choose to assay the enzyme (protein kinase)

A

They used modified inactive PKB to decree it phosphorylation by the PK

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26
Q

In case study 1 (ion exchange) what was the source tissue
And why

A

Rabbit skeletal muscle

Good to get in bulk and purify endogenous factors from

The muscle is insulin responsive tissue so it has all the thing needed to do the signalling events

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27
Q

What is the result of activated PKB

A

protein synthesis

Glucose uptake

Glycogen synthesis

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28
Q

What are the three insulin responsive tissues in the body

A

Skeletal muscle

Adipose tissue

Hepatocytes

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29
Q

What is the pathway of activating PKB

What is the researchers not know

A

In response to insulin, PIP3 gets activated and phosphorylates PDK1 to activate it

PDK1 phosphorylates PKB to activate PKB

PKB causes protein synthesis, glucose uptake, and glycogen synthesis

Didn’t know what that PDK1 is what phosphorylates PKB

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30
Q

What did the researches put in the assay of PKB and the kinase that activates it (case study 1)

A

They had a tagged inactive PKB (GST-PKB)

Buffer

Mg-atp

And the PIP3 lipid

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31
Q

Why was mg-atp in the case study 1 PKB assay

A

Because the PK needs a phosphoryl donor to drive the phosphorylation reaction

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32
Q

What is heparin sepharose

A

It’s negatively charged carbohydrate

function as a cation exchanger

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33
Q

In the method to purify PDK1 (case study 1) what was the tris-HCL , EDTA, EGTA, NAF, PMSF/BENZAMADINE, and 2-ME in the lysis buffer for

A

TrisHCL : too keep at 7.5 ph (need more than 5mM, usually 25mM

EDTA Chelates heavy metal, EGTA CHALATES CA, this block protease since protease need these

NaF fluoride blocks phosphotases (want to block phosphotase to keep our enzyme phosphorylated and active so we can do the experiment)

PMSF/BENZAMADINE, and 2-ME are protease inhibitors

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34
Q

What does 2.5 volumes mean

A

W/V

So per 1g/2.5 volumes

So total 1250 mL

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35
Q

When centrifuging the skeletal muscle in case study 1, what is in the pellet

A

The insoluble myofibrils

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36
Q

In case study 1, why was the supernatent poured through a Büchner funnel with QAE in buffer c

What does this help with

A

This is a capture step

The proteins with the proper charge bind to the QAE while the rest gets filtered through

This help process a large sample and do the wash and elution steps quickly

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37
Q

In case study 1, when washing the QAE funnel column why did they go from 50mM-200mM salt to elute

What is the step bump

A

The wash with 50mM got rid of the flow through

The 200mM was what they needed to elute

Did a step bump where instead of running a gradient they go from low to a high concentration to elute

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38
Q

In case study 1, when washing the QAE funnel column why did they go from 50mM-200mM salt to elute

What is the step bump

A

The wash with 50mM got rid of the flow through

The 200mM was what they needed to elute

Did a step bump where instead of running a gradient they go from low to a high concentration to elute

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39
Q

If it’s say 50% by mass of something what does it mean

A

50% w/v

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40
Q

How do you calculate the ml of matrix

A

Volume= pi r^2 h

If it’s an 11 x 1.6cm column

R= 1/2 (1.6)

H= 11

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41
Q

In case study 1, after getting a pellet from peg fractionation, why did there filter the resuspended pellet

A

There could still be insoluble protein in the resuspended pellet

Don’t want that to clog the column

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42
Q

In case study 1, how did they create the linear salt gradient

A

Through an FPLC system

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43
Q

Case study 1: After Resuspending the peg fractionation pellet, why did the add the sample directly onto the IEX column without dializaying

A

They found the the protein stil doesn’t not elute at 0.1M (which is what the column was equillibrated in)

so they can just add it straight cause they know it won’t elute, this saves time

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44
Q

In case study 1 why did they concentrate the pooled fractions and then just dilute them again

A

When they concentrate it, the protein is concentrated but the salt concentration stays the same

Then when diluting it, the salt concentration decreases

This is a way to concentrate the protein but also lose salt for the next ion exchange column step

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45
Q

In case study 1 why did they use a MgCl2 gradient instead of Nacl for the last ion exchange

A

In the active site of of the PK, mg is used for its activity

So They fractionate differently if you use mgcl vs nacl

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46
Q

What is a common way to store pooled fractions from a chromatography

A

Snap freeze them in liquid nitrogen store at -80

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47
Q

In case study 1 If there is no pip3 in the assay what happens to the activity

A

The activity is zero because pip is needed to activate the enzyme PDK

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48
Q

In case study 1 two peaks with activity show up, give reasons why the first peak I showing in IEX

A

The single PDK is needed for activity, but a protease could have cleave that PDK

Now the charge of the two things are diff (so they elute at diff times)

But both parts are still active

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49
Q

If you run each fraction from IEX peak on an sds page in order what would you see

A

You’d see that the protein of interest shows up in the fraction with the highest activity

You can cut that peice out and do more assays with it

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50
Q

What is size exclusion/gel filtration chromatography

What elutes first

What special about what elutes last

A

Separation of things with diff size and shape through a bead matrix

Bigger elute first, smaller elute last

Smaller things that elute last could also just be salt ions (since salt small)

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51
Q

What can size exclusion / GF (SE/GF) be used for

A

Desalt

To purify

To check if there is complex formation

To see the native mass of the protein (or complex)

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52
Q

What can size exclusion / GF (SE/GF) be used from

A

Desalt

To purify

To check if there is complex formation

To see the native mass of the protein (or complex)

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53
Q

Why does SEC work best if the sample size is small

A

If the sample is small, you get better separation between each sample since it’s not a huge amount of the column all at once

If big, there’s a lot all at once and there isn’t enough space between the beads for good separation (since sample is already in those spaces)

54
Q

Why does band broadening happen in SEC

A

If sample spends more time interaction with the beads in the column

It spreads up in the column so the band also spreads out

55
Q

If the column for SEC is badly packed what happens

A

The sample flows unevenly through the flow adaptor and as a result there is not good separated of peaks

56
Q

What is special about the pores in SEC

A

Can be manufactured to have diff pore sizes to fractionate specific proteins better in a column

57
Q

Does SEC give lots of purification?

What’s the exception

A

No only moderate

Unless the protein is very small or very large then yes is purified it well

58
Q

Why is SEC usually at the end of purification procedure and after IEX

A

Because of the buffer used in it

The buffer is 150mM salt meaning we don’t have to get rid of the salt from the IEX step and can go straight to SEC

59
Q

Why is 150mM salt an important number

A

This is the physiological ionic strength in the cell

60
Q

What is the most importantly thing SEC tells us

A

The oligomeric state of the protien

Ex. Dimer or trimer

61
Q

In sec What is void (V0) and elution volume (Ve)

A

Void volume is the volume of buffer that fills the space outside of the beads

Also the volume it takes for the biggest thing to elute out of the column

Elution volume: the column it takes to the samples to elute out of the column

62
Q

What do we use to determine the V0 in SEC

A

Blue dextran

63
Q

In using SEC for purification what makes it not as useful

what does a sharp and symmetric peak mean

What does a smaller sample mean

A

Most proteins fit in the size range of our protein

The means the protien is pure

You have better resolution of the peak

64
Q

How can SEC be used to desalt our protein

A

Since salt is small it elutes slower than our sample

Then we can collect the fractions with the sample and discard the salt

65
Q

How can SEC be use to determine the mass of your protein

A

You can run standards and see which standard maps your protein matches up with

66
Q

In using SEC to determine the mass of your protein

if the predicted mass of your protein is 1kda and it matches a mass of 2 during SEC

What does this mean

A

The protien likely forms a dimer with itself and the predicted mass it showing the mass when it’s not a dimer

67
Q

How can SEC be used to demonstrate complex formation

A

Run one protein and another protein separately in SEC

Then mix two proteins together in solution and put them in a SEC

68
Q

If you do a SEC to check for complex formation and find that the sample with just one protein that supposed to be 5kda is actually 10 what does this mean

But the band that’s supposed to be one protein at 2kda is actually 1.5

A

That protein form a dimer with itself (since it double)

That protein may have a different shape (rod shape makes it seem smaller in GF meaning it travels slower and has lower observed MW)

That rod shaped one it still a monomer

69
Q

If you do a SEC to check for complex formation and find that the mixed solution has the added witchy of the dimer and the single minor what does this mean

A

The dimer and monomer form a complex with each other

70
Q

For SEC In the equation of finding Kav (MW of protein)

What is Ve, V0,Vt,

How do you find each of these values

A

Ve is the proteins elution volume

V0 is the void volume (the elution volumn of blue dextran)

Vt is the total bed volume (by doing v= pi r^2 h)

Take the volume at the centre of each peak

71
Q

For SEC In the equation of finding Kav (MW of protein)

What is Ve, V0,Vt,

How do you find each of these values

A

Ve is the proteins elution volume

V0 is the void volume (the elution volumn of blue dextran)

Vt is the total bed volume (by doing v= pi r^2 h)

Take the volume at the centre of each peak

72
Q

if for SEC they give the column as 10/300 GL what does this mean

A

The dimensions are 10mm x 300mm

73
Q

Way to improve purification ion IEX do same ph

A

Slide 123

74
Q

What is a superdex particle

Why would we use it

A

It’s a bead of cross linked agarose with dextran in it

It has a specific range of molecules that it can separate so we can use this to separate a protein if we know it’s MW

75
Q

Slide 138

A

What do we need to know

76
Q

What are the three types of muscles

A

Skeletal (striated)

Cardiac (more like skeletal than smooth)

Smooth

77
Q

What is smooth miscle

A

Involuntary miscle, lines the blood vessels and intestines

Involved in blood pressure

78
Q

What is there to know about The ferritin and cyt c standard in SEC

A

They show three peaks, the cuyt c , ferritin, and a aggregate peak at the very biggining

79
Q

In case study 2 what were they purifying, why

A

The myosin phosphotase in smooth muscle

Knew that it was different from the enzyme in the skeletal muscle, but wanted to know more

80
Q

What two things are involved in muscle contraction

A

Actin and myosin

81
Q

What causes contraction of the muscles

Relaxation

A

When the myosin light chain is phosphorylated, contraction

Dephospohrylated: relaxation

82
Q

In case study 1 what is the source tissue and why

Why didn’t they use smooth muscle from the small intestine

A

Chicken gizzards

They are a good source of smooth muscle

Not small intestine because it’s more complex and has a lot of other proteins attached

83
Q

In case study 2 of the myosin phosphotase, how did they get the substrate and do the assay

A

The MLCK phosphorylates the MLC using radioactive phosphate (32P) from ATP

So they let this happen to get 32p-MLC and dialyzed the sample to remove excess atp

Then they assay the 32p-MLC with the fraction that come from the chromatography

84
Q

In case study 2, How does the phosphotase release the radioactive phosphate from 32p-MLC

And how do we know what fraction has the phosphotase

A

It uses water

we check each column fraction for how well they de phos the mlc

85
Q

What’s the difference in DEAE and Q sepharose

A

They’re both anion exhange but they have diff functional groups

86
Q

What is more abundant: PDK1 in skeletal muscle or Myosin phosphotase in smooth muscle

How do we know

A

Mysosin phosphotase

We know bcause once purified, there is more mg of the phosphotase than the PKD1

87
Q

As you continue to purify your protein , what should be happening to the activity and why

A

Activity should be going down because the enzymes starts to die

88
Q

As you continue to purify your protein , what should be happening to the activity and why

A

Activity should be going down because the enzymes starts to die

89
Q

What should you look at to see if the purification was good

A

The specific activity , food purification, yields %

90
Q

What should you look at to see if the purification was good

A

The specific activity , food purification, yields %

91
Q

In case study 2 (phosphotase) why did they reblend the myofibril pellet

A

They found that the phosphotase was actually bound to the myofibrils

Meaning the phosphotase was actually in the pellet

So they blended the pellet to separate the phosphotase

92
Q

In case study 2 What actually released the phosphotase from the myofibrils

A

The buffer that had the detergent + 0.6M NaCl

93
Q

In case study 2, why did they diluted the myofibril pellet solution that has the separated phosphotase and the myofibrils

A

The solution was diluted 2 fold (from 0.6M NaCl to 0.3)

This made the soluble myofibrils come back out of the solution and left the phosphotase out of the solution

94
Q

What is PEG useful for

A

Reducing volume

Removing salt

Purifcation

95
Q

What is Brij 35

A

A mild detergent

96
Q

What is a step bump

A

They test at what point salt concentration the protein elutes and go directly to that concentration to elute on IEX

97
Q

In case study 1 How did they store the solution

What is they then have to do when un storing it

A

They dialyzed it with glycerol and froze it (kept it more stable)

Had to dilute it to remove the glycerol

98
Q

In case study 2 why did they concentrate the solution to a small volume before doing gel filtration

A

To get better peaks because gel filtration works best with smaller samples

99
Q

If a 2 fold purification effective?

A

It’s fine, if we did not do a certain step that gave small purification, some protein that got removed in that step may not have been removed in the other steps

100
Q

In a chromtogrsphy profile, if we see that the a280 is really big in the early fractions what does this mean

A

We got rid of a lot of contaminating proteins

101
Q

How do you know if the enzyme you have is stichiometric based on it being in SDS page

A

If they were in a 1:1 molar ratio

It would show multiple bands each at the same ratio of MW apart

You would also get double the signal intensity

Ex. Bands at 130 , 37, 20 are the same ratio apart and would have most intenstity at the top least at the bottom

102
Q

What is hydrophobic interaction chromatography

What inhancs the interaction

A

Separation based on the interaction between a protein and the hydrophobic surface

By high ionic strength buffer (so they bind more tightly when high salt)

103
Q

What is elution based on in HIC

A

The amount of Hydrophobicity

More hydrophobic, more they don’t like the salt, tighter they bind

104
Q

What is the salt gradient in HIC

A

High salt to low salt

105
Q

Other than decreasing salt to elute what can we use in HIC

Why

A

Increasing chaotropic agent

This disrupts the hydrophobic interactions of the molecules with the column

If the molecule doesn’t come off from the salt

106
Q

In HIC how many elution buffers could we have

A

Buffer a : with salt decreasing

Buffer B with increasing ethylene glycol (chaotropic agent)

107
Q

What is the name for the chaotropic agent

A

Ethylene glycol/ethanediol

108
Q

If your adding ammonium sulfate to the HIC column to protons the binding to the column, what do you have to make sure

How would you make sure of this

A

Make sure that you don’t add too much which makes the protein precipitate out

Add it, stir, centrifuge, see if protein is still in the soluble phase

109
Q

What is the salt content of the start and elution buffer

A

Start is no salt

Elutiong has salt

110
Q

What are the most common hydrophobic ligands that are used in HIC

A

In increasing Hydrophobicity ;

Butyl
Phenyl
Octyl

111
Q

Superose column is a

A

Gel filtration

112
Q

In SDS if something is a dimer how would it show

A

There would be two bands IN EQUAL RATIO of intensity

113
Q

In SDS if something is a dimer how would it show

A

There would be two bands IN EQUAL RATIO of intensity

114
Q

I you see mutiple bands of activity in single different fractions on SDS page and they are not due to contaminant’s what can you assume

A

These bands are all due to the protein

But they may be different proteoforms in each fraction causing different bands sizes:

Could be Multiple genes that make related proteins that all have that same activity which we see

Single gene product, and a protease chopped it up to change its size but keep its activity

Single protein with a PTM that can change its mass

Single gene could give rise to different protein product size through alternative splicing but same activity.

115
Q

What do you not do to your protein if youve purified it

A

Don’t freeze thaw because it can get rid of activity of the protein

116
Q

What do you do to your protein if youve purified it

A

Keep refrigerated at 4 degrees in a closed vessel (to minimize bacterial growth)

Store it as a precipitate in high concentration of ammonium sulfate (4M)

Freeze in 50% glycerol

Add stabilizing agent like glycerol and serum and serum albumin to keep activity of the enzyme

117
Q

What did they want to do in case study 3, what was the source tissue, how did they assay it

A

Wanted to purify PEPC (phosphoenol pyruvate carboxylase) because it’s important in plant metabolism

They did a coupled reaction with porcine (pig) malate dehydrogenase to see the oxidation of NADH at 340nm to see activity of PEPC

Source was arabadopsis Thalia a (plant) cells

118
Q

How does the couple assay for PEPC work

A

PEPC turns PEP into oxaloacetate (in Krebs cycle)

MDH (malate dehydrogenase) turns oxaloacetate to Maltate by oxidizing NADH

So by seeing how much NADH got oxidized at 340nm, we can see how much oxaloacetate PEPC made

119
Q

In case study 3 why did they have a buffer with no salt but with elthylene glycol

A

Used a a chaotropic agent so they can elute the protein of salt doesn’t work

120
Q

In case study 3 why did they have a buffer with malate

A

The malate is an allosteric regulator of the PEPC

It binds to a diff site than the active site of the enzyme

It actually stabilizes it

121
Q

What is Butylspeharose

DEAE

superdex

Mono Q

A

HIC
AIEX
SEC
AIEX

122
Q

If you have a high fold purification is it a high or low abundance protein

A

High abundance

123
Q

In case study 3 why did they have a buffer with imidazole

A

Idk

124
Q

In case study 3 why did they concentrate to 3 mL and dilute to 20mL before IEX

A

Concentrating isn’t keeps the same salt concentration but diluting it after removes the salt so they can do the IEX

20/3 fold dilution

125
Q

Why did they have such a high KDa cutoff size for the vivaspin concentrator

A

To get the biggest molecules out of solution and have it very concentrated

126
Q

In case study 3 After using buffer e to equailitate the column and in the sample why didn’t they dialyze before doing IEX

A

Because v muffler e didn’t have salt in it so they had no salt they needed to remove

127
Q

If you have tiny sample sizes due you have to worry about freeze thawing effects

A

No because it all gets used up in each experiment so you don’t have to restore it

128
Q

What is the typical gradient in cv to use in IEX

A

A gradient over 10CV

129
Q

If we have 2 IEX done at the same ph, how can we improve the separation

A

Use a shallower salt gradient (increases slower so that more things have more times to elute and separate).

130
Q

If we have 2 IEX done at the same ph on the same column how can we improve the separation

A

Use a shallower salt gradient (increases slower so that more things have more times to elute and separate)

when doing this you stretch it over longer column volumes

Now all the proteins that used to come out together are now fractionation at diff points

131
Q

If we have 2 IEX done at the the same column and same gradient how can we improve the separation

A

If anion exchange , Shift the pH to a higher pH, by dialyzing at a higher pH

This causes some proteins to fractionate away from each other based on the charge changing

132
Q

What are two ways to get better separation in IEX

A

Shift the ph

Run a shallower gradient

Bchem 411 (9 decks)

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