Part II 100-150 Flashcards
(132 cards)
1
Q
What is ion exchange chromatography
A
separation based on the reversible interaction between a charged protein and and oppositely charged chromatographic medium
2
Q
In a cation exhange chromatography what elutes later
A
More postive elites later since more tightly bound
3
Q
If the matrix is positive charged in ion exchange what type ion exchange is it
A
Anion exhange
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
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
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
7
Q
What comes out of the ion exhange column right after loading it
A
The unbound molecules (flowthrough)
8
Q
What is the salt gradient in ion exchange chromatography
A
Low to high
9
Q
What pH is cation exchange generally run at
Anion
A
Low pH (5.5-6)
Higher pH (8-8.5)
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
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
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
13
Q
In ion exchange What does it mean when the ph = pi
A
The net charge of the protein is zero
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)
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)
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
17
Q
What does strong ion exchanger mean
A
If means the column is fully charged over a greater pH range
18
Q
What does DEAE stand for
A
Diethylaminoethyl
19
Q
I’m ion exchange chromatography what does the A280 monitor
A
20
Q
I’m ion exchange chromatography what does the A280 monitor
A
The concentration of protein in the fraction in mg/ml
21
Q
What happens to the A280 if you have a really put protein
A
The a280 corresponds (lines up) to the activity peak
22
Q
If there is a diagram on the chromatography profile what do we call it
A
An inset
23
Q
What is considered a shallow salt gradient in ion exchange chromatography
A
0-200mM
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
25
In case study 1 (ion exchange) how did they choose to assay the enzyme (protein kinase)
They used modified inactive PKB to decree it phosphorylation by the PK
26
In case study 1 (ion exchange) what was the source tissue
And why
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
27
What is the result of activated PKB
protein synthesis
Glucose uptake
Glycogen synthesis
28
What are the three insulin responsive tissues in the body
Skeletal muscle
Adipose tissue
Hepatocytes
29
What is the pathway of activating PKB
What is the researchers not know
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
30
What did the researches put in the assay of PKB and the kinase that activates it (case study 1)
They had a tagged inactive PKB (GST-PKB)
Buffer
Mg-atp
And the PIP3 lipid
31
Why was mg-atp in the case study 1 PKB assay
Because the PK needs a phosphoryl donor to drive the phosphorylation reaction
32
What is heparin sepharose
It’s negatively charged carbohydrate
function as a cation exchanger
33
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
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
34
What does 2.5 volumes mean
W/V
So per 1g/2.5 volumes
So total 1250 mL
35
When centrifuging the skeletal muscle in case study 1, what is in the pellet
The insoluble myofibrils
36
In case study 1, why was the supernatent poured through a Büchner funnel with QAE in buffer c
What does this help with
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
37
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
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
38
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
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
39
If it’s say 50% by mass of something what does it mean
50% w/v
40
How do you calculate the ml of matrix
Volume= pi r^2 h
If it’s an 11 x 1.6cm column
R= 1/2 (1.6)
H= 11
41
In case study 1, after getting a pellet from peg fractionation, why did there filter the resuspended pellet
There could still be insoluble protein in the resuspended pellet
Don’t want that to clog the column
42
In case study 1, how did they create the linear salt gradient
Through an FPLC system
43
Case study 1: After Resuspending the peg fractionation pellet, why did the add the sample directly onto the IEX column without dializaying
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
44
In case study 1 why did they concentrate the pooled fractions and then just dilute them again
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
45
In case study 1 why did they use a MgCl2 gradient instead of Nacl for the last ion exchange
In the active site of of the PK, mg is used for its activity
So They fractionate differently if you use mgcl vs nacl
46
What is a common way to store pooled fractions from a chromatography
Snap freeze them in liquid nitrogen store at -80
47
In case study 1 If there is no pip3 in the assay what happens to the activity
The activity is zero because pip is needed to activate the enzyme PDK
48
In case study 1 two peaks with activity show up, give reasons why the first peak I showing in IEX
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
49
If you run each fraction from IEX peak on an sds page in order what would you see
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
50
What is size exclusion/gel filtration chromatography
What elutes first
What special about what elutes last
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)
51
What can size exclusion / GF (SE/GF) be used for
Desalt
To purify
To check if there is complex formation
To see the native mass of the protein (or complex)
52
What can size exclusion / GF (SE/GF) be used from
Desalt
To purify
To check if there is complex formation
To see the native mass of the protein (or complex)
53
Why does SEC work best if the sample size is small
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
Why does band broadening happen in SEC
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
If the column for SEC is badly packed what happens
The sample flows unevenly through the flow adaptor and as a result there is not good separated of peaks
56
What is special about the pores in SEC
Can be manufactured to have diff pore sizes to fractionate specific proteins better in a column
57
Does SEC give lots of purification?
What’s the exception
No only moderate
Unless the protein is very small or very large then yes is purified it well
58
Why is SEC usually at the end of purification procedure and after IEX
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
Why is 150mM salt an important number
This is the physiological ionic strength in the cell
60
What is the most importantly thing SEC tells us
The oligomeric state of the protien
Ex. Dimer or trimer
61
In sec What is void (V0) and elution volume (Ve)
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
What do we use to determine the V0 in SEC
Blue dextran
63
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
Most proteins fit in the size range of our protein
The means the protien is pure
You have better resolution of the peak
64
How can SEC be used to desalt our protein
Since salt is small it elutes slower than our sample
Then we can collect the fractions with the sample and discard the salt
65
How can SEC be use to determine the mass of your protein
You can run standards and see which standard maps your protein matches up with
66
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
The protien likely forms a dimer with itself and the predicted mass it showing the mass when it’s not a dimer
67
How can SEC be used to demonstrate complex formation
Run one protein and another protein separately in SEC
Then mix two proteins together in solution and put them in a SEC
68
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
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
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
The dimer and monomer form a complex with each other
70
For SEC In the equation of finding Kav (MW of protein)
What is Ve, V0,Vt,
How do you find each of these values
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
For SEC In the equation of finding Kav (MW of protein)
What is Ve, V0,Vt,
How do you find each of these values
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
if for SEC they give the column as 10/300 GL what does this mean
The dimensions are 10mm x 300mm
73
Way to improve purification ion IEX do same ph
Slide 123
74
What is a superdex particle
Why would we use it
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
Slide 138
What do we need to know
76
What are the three types of muscles
Skeletal (striated)
Cardiac (more like skeletal than smooth)
Smooth
77
What is smooth miscle
Involuntary miscle, lines the blood vessels and intestines
Involved in blood pressure
78
What is there to know about The ferritin and cyt c standard in SEC
They show three peaks, the cuyt c , ferritin, and a aggregate peak at the very biggining
79
In case study 2 what were they purifying, why
The myosin phosphotase in smooth muscle
Knew that it was different from the enzyme in the skeletal muscle, but wanted to know more
80
What two things are involved in muscle contraction
Actin and myosin
81
What causes contraction of the muscles
Relaxation
When the myosin light chain is phosphorylated, contraction
Dephospohrylated: relaxation
82
In case study 1 what is the source tissue and why
Why didn’t they use smooth muscle from the small intestine
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
In case study 2 of the myosin phosphotase, how did they get the substrate and do the assay
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
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
It uses water
we check each column fraction for how well they de phos the mlc
85
What’s the difference in DEAE and Q sepharose
They’re both anion exhange but they have diff functional groups
86
What is more abundant: PDK1 in skeletal muscle or Myosin phosphotase in smooth muscle
How do we know
Mysosin phosphotase
We know bcause once purified, there is more mg of the phosphotase than the PKD1
87
As you continue to purify your protein , what should be happening to the activity and why
Activity should be going down because the enzymes starts to die
88
As you continue to purify your protein , what should be happening to the activity and why
Activity should be going down because the enzymes starts to die
89
What should you look at to see if the purification was good
The specific activity , food purification, yields %
90
What should you look at to see if the purification was good
The specific activity , food purification, yields %
91
In case study 2 (phosphotase) why did they reblend the myofibril pellet
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
In case study 2 What actually released the phosphotase from the myofibrils
The buffer that had the detergent + 0.6M NaCl
93
In case study 2, why did they diluted the myofibril pellet solution that has the separated phosphotase and the myofibrils
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
What is PEG useful for
Reducing volume
Removing salt
Purifcation
95
What is Brij 35
A mild detergent
96
What is a step bump
They test at what point salt concentration the protein elutes and go directly to that concentration to elute on IEX
97
In case study 1 How did they store the solution
What is they then have to do when un storing it
They dialyzed it with glycerol and froze it (kept it more stable)
Had to dilute it to remove the glycerol
98
In case study 2 why did they concentrate the solution to a small volume before doing gel filtration
To get better peaks because gel filtration works best with smaller samples
99
If a 2 fold purification effective?
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
In a chromtogrsphy profile, if we see that the a280 is really big in the early fractions what does this mean
We got rid of a lot of contaminating proteins
101
How do you know if the enzyme you have is stichiometric based on it being in SDS page
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
What is hydrophobic interaction chromatography
What inhancs the interaction
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
What is elution based on in HIC
The amount of Hydrophobicity
More hydrophobic, more they don’t like the salt, tighter they bind
104
What is the salt gradient in HIC
High salt to low salt
105
Other than decreasing salt to elute what can we use in HIC
Why
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
In HIC how many elution buffers could we have
Buffer a : with salt decreasing
Buffer B with increasing ethylene glycol (chaotropic agent)
107
What is the name for the chaotropic agent
Ethylene glycol/ethanediol
108
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
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
What is the salt content of the start and elution buffer
Start is no salt
Elutiong has salt
110
What are the most common hydrophobic ligands that are used in HIC
In increasing Hydrophobicity ;
Butyl
Phenyl
Octyl
111
Superose column is a
Gel filtration
112
In SDS if something is a dimer how would it show
There would be two bands IN EQUAL RATIO of intensity
113
In SDS if something is a dimer how would it show
There would be two bands IN EQUAL RATIO of intensity
114
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
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
What do you not do to your protein if youve purified it
Don’t freeze thaw because it can get rid of activity of the protein
116
What do you do to your protein if youve purified it
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
What did they want to do in case study 3, what was the source tissue, how did they assay it
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
How does the couple assay for PEPC work
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
In case study 3 why did they have a buffer with no salt but with elthylene glycol
Used a a chaotropic agent so they can elute the protein of salt doesn’t work
120
In case study 3 why did they have a buffer with malate
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
What is Butylspeharose
DEAE
superdex
Mono Q
HIC
AIEX
SEC
AIEX
122
If you have a high fold purification is it a high or low abundance protein
High abundance
123
In case study 3 why did they have a buffer with imidazole
Idk
124
In case study 3 why did they concentrate to 3 mL and dilute to 20mL before IEX
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
Why did they have such a high KDa cutoff size for the vivaspin concentrator
To get the biggest molecules out of solution and have it very concentrated
126
In case study 3 After using buffer e to equailitate the column and in the sample why didn’t they dialyze before doing IEX
Because v muffler e didn’t have salt in it so they had no salt they needed to remove
127
If you have tiny sample sizes due you have to worry about freeze thawing effects
No because it all gets used up in each experiment so you don’t have to restore it
128
What is the typical gradient in cv to use in IEX
A gradient over 10CV
129
If we have 2 IEX done at the same ph, how can we improve the separation
Use a shallower salt gradient (increases slower so that more things have more times to elute and separate).
130
If we have 2 IEX done at the same ph on the same column how can we improve the separation
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
If we have 2 IEX done at the the same column and same gradient how can we improve the separation
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
What are two ways to get better separation in IEX
Shift the ph
Run a shallower gradient
