Marie Ange
PREPARATION OF
THE SINGLE AND DOUBLE
MUTANTS OF FecA
1
Cysteine Mutant - QuickChange Site-Directed Mutagenesis
Kit (stratagene)
1. Materials Provided
2. Storage Conditions
XL1-Blue supercompetent cells: –80°C  upstairs
All Other Components: –20°C freezer  upstairs
3. Method Overview
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4. Primer design Guide lines
The mutagenic oligonucleotide primers for use in this protocol must be designed
individually according to the desired mutation. The following considerations should be
made for designing mutagenic primers:
Helpful website for designing primers : you can check the secondary structure and dimer
formation of primers!
http://www.sigma-genosys.com/calc/DNACalc.asp
* Both of the mutagenic primers must contain the desired mutation and anneal to the
same sequence on opposite strands of the plasmid.
* Primers should be between 25 and 45 bases in length, and the melting temperature
(Tm) of the primers should be greater than or equal to 78°C.
The following formula is commonly used for estimating the Tm of primers:
For calculating Tm:
Tm = 81.5 + 0.41(%GC) - 675/N - %mismatch
• N is the primer length in bases
• values for %GC and % mismatch are whole numbers
For calculating Tm for primers intended to introduce insertions or deletions, use this
modified version of the above formula:
Tm = 81.5 + 0.41(%GC) - 675/N
Where N does not include the bases which are being inserted or deleted.
* The desired mutation (deletion or insertion) should be in the middle of the primer with
~10–15 bases of correct sequence on both sides.
* The primers optimally should have a minimum GC content of 40% and should
terminate in one or more C or G bases.
After you design the primers, you can order them from integrated DNA technologies. (it
takes 2-3 days)
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fill out the LPO form with item description and cost (to get information on cost,
we might need to go ahead to order through online and save the order!)
email the form to “Dan Via (dwv8f@virginia.edu)” or drop it off to his office to
get a PO number
order through online and check out with the PO number
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www.idtdna.com and type id : cafisolab and password : rOOm196
Same day oligo service / DNA oligos, One at a time
Scale - 25nmole DNA oligo and Purification – standard desalting
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3
5. Procedure
(highlightened in yellow : you can vary the amount by experience and depending on the
case)
1) Prepare the DNA template
Grow overnight in 6 mL of LB, in order to do 2 miniprep and make sure you have
enough DNA for future work.
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Use 2 eppendorf
Add 1.5 mL of culture into each tube
Spin down using bench centrifuge
Discard the supernatant
Add again 1.5 mL of culture into each tube
Spin down
Discard the supernatant
Resuspend each pellet with 250 uL buffer P1 (then follow Quiagen kit protocol)
….
Use 2 columns
For each column, elute with 50 uL of sterile water
Pool the DNA together
So, at the end, you will have 100 uL DNA coming from 6 mL culture.
2) Determination of DNA concentration
 Take 10 ul of DNA, add 1 mL of water, so the final volume is 1010 uL
 Use the quartz cuvette, and measure the absorbance at 260nm
 Remember that 1 abs = 50 ug DNA / mL
 So to determine the concentration, you need to apply the formulae:
 [DNA]= Abs at 260 * 50 * 1010/10
 (you need to multiply by the dilution factor. Since you add 10 uL into 1 mL, you
need to multiply your concentration by 101)
 So you will finally have a DNA concentration in ug /mL
 From this point, you need to try different conditions to do Quickchange. You can
try with 5 ng, 10 ng, or up to 50 ng of DNA template….and you will sometimes
need to dilute your template DNA with water so that you can pipet an accurate
volume.
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3) Two primers required for one mutant
- make 100pmol/uL stock solution using sterile ddH2O and keep this in the -20 freezer.
(~ 1200 ng/ul)
- before you start mutagenesis, dilute the stock solution 10 times (for example, take
10ul of 100pmol/ul stock solution and add 90uL of sterile ddH2O to make 120ng/ul
stock)
2) PCR reaction mixture
Total
50L
5L
1L
2L
2L
1L
39L
10X reaction buffer
template DNA
primer #1 (120ng/L)  final concentration ~ 240ng/L
primer #2 (120ng/L)  final concentration ~ 240ng/L
dNTPs
ddH2O
And then add 1L Pfuturbo DNA polymerase “at the end after you mix everything!!”
(The enzyme should be kept at -20 all the time)
3) PCR (thermal cycling)
Alternative program name: MYKIT3 (2hr 30 min)
step 1 : 95 °C 30 sec
step 2: 95 °C 30 sec
step 3: 55 °C 1 min
step 4: 68 °C 6 min (1min for 1kB : ours is around 5kB so.. I added one more min.)
go to step 2
repeat 16 times
keep at 4 °C after the entire cycles
4) DpnI digestion
- add 1 L of the DpnI restriction enzyme directly to each amplification reaction
- incubate at 37 degree for 1 hr for the digestion
5) DNA gel
 not necessary, you can skip this.
- 10 L of DNAs (after digestion) + 3 L of sample buffer (the plasmid is around 5kb)
Ex)
L 1
2
3
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6) Transformation of XL1-Blue (-80 freezer) supercompetent cells
 you need to get ice, and turn on water bath upstairs and set the temp at 42
1. Gently thaw the XL1-Blue supercompetent cells on ice. Aliquot 50 µl of the
supercompetent cells to a prechilled 1.5 ml eppendorf tube.
2. Transfer 1 µl (you can add more) of the Dpn I-treated DNA from each reaction to
separate aliquots of the supercompetent cells.
3. Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30
minutes.
4. Heat pulse the transformation reactions for 45 seconds at 42°C
5. Place the reactions on ice for 2 minutes.
6. Add 300 µl of LB media preheated to 42°C
7. Incubate the transformation reactions at 37°C for 1 hour with shaking at 225–250 rpm.
8. Plate the everything of each transformation reaction into the LB/amp plates
9. Incubate the plates at 37°C for overnight (more than 16 hours)
7) Miniculture for the Glycerol stock and miniprep (DNA extraction)
- Pick 1 colony and grow it in 7ml LB/amp for overnight
Next day morning, the media should look cloudy due to the growth of cells.
40% glycerol + B1 : 20 ml of glycerol + 30 ml of ddH2O  autoclave and cool down 
add B1 (0.005 g / 100 ml of 40% glycerol)
A. XL1 blue glycerol stock
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in a 2ml of cryogenic tube, add 900ul of 40% glycerol+B1 stock (autoclaved)
take 900ul of culture and mix it with glyceorol
keep it at -80 C freezer (label with protein, mutant, strain, date, and your
name)
Note: In case you need more DNAs in the future, you can use this XL1blue stock in
order to restreak the plate! And pick a colony  miniculture  DNA extraction using
miniprep!
B. Miniprep (DNA extraction)
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take 3ml of culture and split it into 2*1.5ml eppendorf tubes
spin them down for 1min at 13,000 rpm using microcentrufuge
repeat again and spin down
decant the supernatant and keep the pellet
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follow the QIAGEN miniprep procedure below
(Note: now, there are two tubes for one mutant)
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Resuspend pelleted bacterial cells in a total 250 µl Buffer P1 (fridge) for one
mutant and transfer to a microcentrifuge tube  usually, I add 125 ul of P1 into
each tube, resuspend, and combine them together into one microcentrifuge tube
Ensure that RNase A has been added to Buffer P1. No cell clumps should be
visible after resuspension of the pellet.
Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
Mix gently by inverting the tube. Do not vortex, as this will result in shearing of
genomic DNA. If necessary, continue inverting the tube until the solution
becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for
more than 5 min.
Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube
4–6 times.
To avoid localized precipitation, mix the solution thoroughly, immediately after
addition of Buffer N3. The solution should become cloudy.
Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
Apply the supernatants from the previous step to the QIAprep spin column by
decanting or pipetting.
Centrifuge for 30–60 s. Discard the flow-through.
Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–
60 s.
Discard the flow-through, and centrifuge for an additional 1 min to remove
residual wash buffer.
Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA,
add 50 µl water to the center of each QIAprep spin column, let stand for 1 min,
and centrifuge for 1 min.
Because we have 2 columns, we can pool 2*50 uL together, so we will have 100
uL of DNA coming from 6 mL of culture.
Throw out the spin column and keep the DNAs at -20 freezer (label with protein,
mutant, strain, date, and your name)
8) DNA Sequencing
Note : now you have 100ul of plasmid DNA
Sequencing company : www.genewiz.com
1) You need to get a PO number from Danny for Genewiz! Now, you need to fill out
the form first with cost and description to get a number!
2) Make an ID in genewiz for sample submission
3) Order through online and print out the order submission form from the website
4) Prepare the samples
In 8-strip PCR tubes,
- 7.5 µl of plasmid DNA
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- 2.5 µl of ddH2O
- 2.5 µl of sequencing primer (from 100 pmol/µl stock solution)
5) Place the DNA in an envelop and go to Mayo’s lab in Jordan Hall and put the
envelop in the Drop Box
6) You will get an email from Genewiz about the results and then you can check the
sequencing results from genewiz website
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It sequence is not correct, throw the glyceol stock and DNA away and go back
to step 7)
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If sequence is correct, do the final transformation into BL21 OmpA-strain (FhuA,
FecA)  proceed to step 9)
9) Final DNA Transformation for growing
In order to make competent cells
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Grow RK5016 or BL21 OmpA- strain directly using a glycerol stock in ~ml of LB (no
antibiotic) (1ml needed for the transformation of 1 mutant) at 34C (RK5016) or
37C (BL21 OmpA-) with 250rpm shaking for 5-6 hours (until it’s in the mid-log
phase)
For 1 mutant,
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Aliquot 1ml of cell culture into 1.5 ml eppendorf tube
Spin it down for 1 min (> 8,000 rpm)
Decant the supernatant and Keep the pellet
Add 30 µl of LB (no amp) and Resuspend the pellet
Mix them well with pipette
Leave on ice for 10 min
Add 50 µl of TSS solution
Mix
Leave on ice for 15 min
** TSS solution (transformation and storage solution)
In 100mL
2g tryptone
1g yeast extract
1g NaCl
2g MgCl26H2O
20g PEG 3350 (PEG 8000 would be ok)
10ml DMSO
Water up to 100ml
pH = 6.5 (should be close already)
 filter sterilize
 aliquot’
 keep it in the fridge (4 degrees)
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Note:
Now, “competent cells are made”
You can freeze these at -80 for future use or proceed to the following step for fresh
transformation.
Final Transformation
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Add 1 µl of DNA into competent cells
Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30
minutes.
Heat pulse the transformation reactions for 45 seconds at 42°C
Place the reactions on ice for 2 minutes.
Add 500 µl of LB media
Incubate the transformation reactions at 37°C for 1 hour with shaking at 225–250 rpm.
Plate the 50-200 µl of each transformation reaction into the LB/amp plates
Incubate the plates at 37°C for overnight (more than 16 hours)
10) Final glycerol stock
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pick one colony and grow it in LB+amp ( > 1ml) overnight
make a glycerol stock (900ul of cells + 900ul glycerol) and keep it at -80
Note:
 BL21 OmpA- strain is not that stable as RK5016 to keep the plasmids. People
usually say BL21 is not safe. So I do a fresh transformation each time
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Growing and Preparing Outer Membranes. I usually grow 2
liters for one mutant….
LB media 1L in 2L flask
10g tryptone
5g yeast extract
10g NaCl
Prepare LB media and autoclave!
Ampicillin stock solution = 1g of amp / 10ml ddH2O. Filter-sterilize! (Ampicillin powder:
cold room)
IPTG 200mM stock solution: 0.72 g IPTG/ 15mL ddH2O) Filter sterilize
1st day
*** Preculture
Use 10 ml of LB and 10 ul of ampicillin per mutant!
Use 50 ml conical tubes for preculture.
You pick a colony from BL21 OmpA- fresh transformation to inoculate and let grow
overnight at 37 degrees!
*** Mainculture
You need 1L of LB per mutant! Add 1ml of ampicillin and add 10ml of preculture to 1L
of LB.
In the morning, inoculate one flask containing 1L of LB with 5 mL of preculture. After 56 hours, it will become cloudy. Then, you can start induction.
*** IPTG induction
I use 3 ml of 200 mM stock solution for one liter culture, the final IPTG concentration in
1L of LB will be 0.6 mM.
Induce when the O.D. reaches ~ 0.8 to 1. Usually, it takes 2-3 hours after you start
the mainculture. Add 3mL IPTG 200 mM solution into one flask to induce protein
expression.
Decrease the temp down to 25 degrees and grow it for 4 hours
Spin down using SL3000 rotor and freeze the pellet.
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PMSF
17 mg/1 ml isoproponal
Resuspension buffer
(10 mM HEPES)
5 % Sarkosyl
2.383 g HEPES / 1 L ddH2O
(Protease inhibitor) keep it in the 20 freezer
Adjust pH to 6.5 and Filter sterilize
12.5 g / 250 ml
(Detergent) Filter sterilize
For 2 L growth!
1) Split 1 L culture into 3 centrifuge tubes
2) Balance with ddH2O. Using the SLA-3000 rotor, spin down the cells for 10 min.
at 6,000 rpm. Discard the supernatant and Keep the pellet!
3) Split the remaining liter into the tubes, and centrifuge again
4) Add 9 ml of resuspension buffer (10 mM HEPES, pH 6.5) to each of the
centrifuge tubes. Resuspend cells by pipetting. You will have more than 27 mL.
Pour all resuspended cells into 2 conical tube and bring volume to 30 ml with
resuspension buffer. So, for 2 liters of culture, you will have 2 canonical tubes
with 30 mL of resuspended bacteria.
5) French press cells passing each lysate through the press twice. After the
second pass, the cells should be collected into a new tube with 50 ul of PMSF.
6) Place the lysed cells into a centrifuge tube and spin using the SS-34 rotor at
10,000 rpm for 10 min. Balance with resuspension buffer. Discard pellet and
keep the supernatant.
7) Place supernatant into 2 of vacuum ultra centrifuge tubes per mutant (Now, we
have ~30 ml of volume but the tube for ultracentrifuge can only hold ~25 ml.
Therefore, we need to split it into two tubes. Each ultracentrifuge tube will have
around 15 mL of supernatant. So now, you have 4 ultracentrifuge tubes). Add
2.5 ml of 5% Sarkosyl into each ultracentrifuge tube. Balance with resuspension
buffer. Spin at 30,000 rpm for 1.5 hr using ultracentrifuge.
8) Discard supernatant. Resuspend pellet with HEPES buffer. Use 2 ml per liter of
culture. Keep the resuspended pellet in the autoclaved eppendorf tubes. Put
them in the -20 degree freezer until solubilization.
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Solubilization
Now, you have outer membranes in the -20 freezer.
You can keep them for a long period.
You need,
1M DTT in HEPES buffer (reconstitution buffer) or sodium acetate
100mM Tris buffer in water (12.11g/1L, pH 8.05) : filter, you don’t need to degas it!
0.125M EDTA : We have 0.5M EDTA solution. Dilute this to make 0.125M.
Let thaw outer membranes at room temperature.
Take out OG (detergent at -20), let it be at room temperature
In each 50 mL conical tube,
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1) ~ 10 mL 100 mM Tris buffer
2) Add 1.6 mL of 0.125 M EDTA
3) 0.5 g OG
4) 6 L 1M DTT or 10 uL DTT for double mutants
5) outer membrane sample corresponding to 1 liter culture
6) Add buffer up to 25 mL
Incubate at room temperature for 3 hrs on shaking gel table
Place the sample in an ultracentrifuge tube
Balance samples with 100 mM Tris buffer (pH 8.05)
Spin down at 25,000 RPM for 1 hr
Keep the supernatant in 50ml conical tube and store it in the cold room (4
C)
Spin labeling for single mutants preparation
I weight 3,3 mg for 500 uL of Ethanol, and add 200 uL of each reagent into each
50 ml canonical tube containing 25 mL of solution.
I let react in the dark Over Night at Room Temp.
Concentration 1 (for double mutants preparation)
Now, you have 2* 25 mL= 50 mL for 2 liters of culture.
Pool the sample together, and concentrate down to 20 mL using the concentrator and
the millipore membrane 10000Da 44.5 mm diameter. The pressure should be around 50
psi.
Spin labeling 1 (for double mutants preparation)
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Split the concentrated sample into 2, to have 10 mL of solubillized outer
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membrane.
I add lots of MTSL here because I added 10 umole of DTT. So I want 2
times more MTSL, in order to have 20 umole MTSL.
Make 100mM MTSL (in -20 freezer, blue box) spin labeling reagent (Dissolve
~ 26 mg of MTSSL in 1000 L absolute EtOH, : measure out MTSL in the
dark!
Add 200 L of MTSL reagent into each 50ml conical tube (1L growth, one
mutant)
React in the dark (use foil or put them in the drawer) at 37 degrees
overnight
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Purification using Anion Exchange (AKTA purifier)
You need,
1) Buffer A (17mM OG, 25mM Bistris, pH 7.0 = for 1L, in 800ml of ddH2O, add 5g
OG and 6.14g of Bistris, adjust pH to 7.0, and then fill water up to 1L)
2) Buffer B (17mM OG, 25mM Bistris, 1M LiCl, pH 7.0 = for 1L, in 800ml of ddH2O,
add 5g OG, 6.14g of Bistris and 42.39g of LiCl, adjust pH to 7.0, and then fill
water up to 1L)
3) 20% ethanol water (200ml of 95% ethanol + 800ml of ddH2O)
4) ddH2O
Make all of them and “FILTER and DEGAS!!!”
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Turn on the purifier
Open the program in computer after the machine is turned on.
Now, all the leads should be in 20% EtOH-water.
(System Control Window)
1. Run pump wash purifier [pump pump wash purifier  execute] on All 4
(A1,A2,B1,B2: ON,ON,ON,ON)
2. Wash all the leads with ddH2O
3. Place all the leads into filtered+degassed ddH2O
4. Run pump wash purifier on All 4 (A1,A2,B1,B2: ON,ON,ON,ON)
5. Load water for 5 min : [Flow path  Position  Load] [Pump  Flow rate 
2ml/min] and execute
6. While you’re doing step 5 (flowing water), put the column on! You need to find
appropriate connectors to install the column. After you put the column on, keep
running water for 5 more minutes or until UV line becomes flat!
7. Stop running water [Pump  Flow rate  0ml/min] and execute.
8. Check the superloop! It should be in ddH2O and clean! It not, you need to
clean it! Now, put the superloop onto the machine and connect the green lines!
9. Eject water left in the superloop [Flow path  Position  Inject] [Pump  Flow
rate  2ml/min] and execute  stop [Pump  Flow rate  0ml/min]
10. Place each lead into the correct container and seal the top with parafilm!!
A1: Buffer A (OG, Bistris, pH 7.0, degassed and filtered)
A2: 20% EtOH-water (degassed and filtered)
B1: Buffer B (LiCl, OG, Bistris, pH 7.0, degassed and filtered)
B2: ddH2O (degassed and filtered)
11. Run pump wash purifier on A2 and B2 [pump pump wash purifier  select A2
and B2  execute] (A1,A2,B1,B2: OFF,ON,OFF,ON)
12. Run pump wash purifier on A1 and B1 [pump pump wash purifier  select A1
and B1  execute] (A1,A2,B1,B2: ON,OFF,ON,OFF)
13. Flow 10ml of Buffer A (100% from A, A1) [Flow path  Position  Load], set
0%B [Pump  Gradient  0% B], select A1 [Flow path  pump A inlet  A1],
Start flow at 2ml/min [Pump  Flow rate  2ml/min]  execute
14. Stop after 5 min [Pump  Flow rate  0ml/min]  execute
15. Flow 10ml of Buffer B (100% from B, B1) [Flow path  Position  Load], set
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0%B [Pump  Gradient  100% B], select B1 [Flow path  pump B inlet 
B1], Start flow at 2ml/min [Pump  Flow rate  2ml/min]  execute
16. Set the position at Load [Flow path  position  LOAD  execute] and start
loading your sample into the superloop. (You have 10 mL of solubilized outer
membrane. Use syringe and filter to load your sample and make sure not to get
bubbles!!)
17. Close the window and Hit END
18. Open Q column program file [File Run  Go to Gail’s folder  Select correct
program for each protein] Use program “Wash for FecA”
19. Check the parameters and type “the amount of the sample in the superloop” in
[Empty loop with (sample vol)]
20. Type the file name
21. Make sure 1) waste container in the back is not overflowing 2) buffers, water,
and ethanol-water levels are enough and 3) fraction collector is set up at the
appropriate position and is filled with enough glass tubes  IMPORTANT!!!
22. Hit “Start”
After the 1st sample (usually it takes less than 2 hours to complete the run)
23. Take out tubes from the fraction collector  label  put them in the cold room
to store!
24. Print the chromatogram in evaluation window
25. Cleaning
A. Eject sample left in the superloop [Flow path  Position  Inject] [Pump
 Flow rate  2ml/min] and execute  stop [Pump  Flow rate 
0ml/min]
B. [Flow path  Position  Load] Load 5ml of filtered and degassed ddH2O by
syringe
C. Eject water [Flow path  Position  Inject] [Pump  Flow rate 
2ml/min] and execute  stop [Pump  Flow rate  0ml/min]
D. Repeat B and C one more time
26. Load the second sample (go to step 17-23)
After you finish all the purifications (after step 25),
27. Clean A1 and B1 leads with ddH2O (Put the leads in the beaker containing ddH2O
and wash or you can use squeeze bottle)
28. Place A1 and B1 leads into ddH2O (B2)
29. Run pump wash purifier on A1 and B1 [pump pump wash purifier  select A1
and B1  execute]
30. Place all the leads into 20% EtOH-water (A2)
31. Run pump wash purifier on all 4 [pump pump wash purifier  select all of
them  execute]
32. Flow around 30ml of 20% EtOH-water through the column to clean and store
the column in ethanol water [Flow path  Position  Load] [Pump  Flow rate
 2ml/min] and execute
33. Take out and clean the superloop with ddH2O while you’re doing step 32, (at the
end, fill the superloop with degassed ddH2O)  put the superloop back to the
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purifier
34. Remove the column from the purifier after flowing 30ml of ethanol-water but
while you’re still doing step 32 not to get bubbles  Put the column back in the
cold room and label haw many times you used for your record!
35. Hit “END” to turn off the UV lamp!!!!! Don’t forget this~~ ^^;
36. Exit the software
37. Turn off the purifier!!
*** Here, check the level of EtOH-water if you have enough!!
After the first purification, the protein usually precipitates from fraction 17 to 26, corresponding to
30-40% buffer B, so there is around 300 to 400 mM LiCl.
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SDS-PAGE Gel Electrophoresis
4-15% gels are normally used! You can use others such as 12.5 or 10%.
FecA is 80kD.
Check the samples which precipitate, but also the 2 fractions preceding the 1st
precipitated sample. You can have some pure protein in fraction 16. You usually have
impurity for fractions 26 and 27.
You need, (Here, turn on the heat block)!!
1) Sample buffer : 950 ul of Laemmli sample buffer + 50 ul of
betamercaptoethanol (in the hood)
2) Standard : in the -20 freezer (biorad broad range standard)
For 1 gel, there are 18 lanes. (one lane is enough for 30 ul)
1. Prepare the samples
in 0.5ml of eppendorf tubes,
1) Sample  15ul of sample + 15 ul of sample buffer
2) Standard  1ul of standard + 15 ul of sample buffer
2. In the gel box, check the buffer level and label! (if the buffer has been used
more than 4-5 times, fill it with fresh 1X buffer  there are 1X and 10X gel
running buffer on the shelf  if we don’t have 1X, dilute 10X to make 1X)
3. Put the gel in the gel box (Don’t forget to remove white tape!)  gels are in the
fridge!
4. Fill the buffer
5. Take out the green comb
6. Heat the samples except for standard!! for 2-3 min at 100 degrees
7. Load the samples!
8. Run at 160-180V for < 1hr
9. Stop the power supplier
10. Put the gel in water and change water 2-3 times -2 * 10 minutes- (or you can
leave it like that overnight) until the blue band at the bottom goes away
11. Stain the gel (1 hr – overnight) using biosafe staining solution on the fridge
12. Destain the gel in water
13. Take a picture of the gel!
14. Throw the gel into “protein gel waste container” in the balance room
Concentration 2 (I skip this step for single mutant prep)
Pool the sample from the 2 purifications together, and concentrate down to 10 mL using
the concentrator and the millipore membrane 10000Da 44.5 mm diameter. The pressure
should be around 50 psi.
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Keep the flow through in the cold room. Sometimes, the membrane is leaking, and the
protein will be in the flow through, so the protein will precipitate.
Spin labeling 2 (I skip this step for single mutant prep)
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Split the concentrated sample into 2, to have 5 mL of purified protein into
each tube.
I concentrate here because I want to decrease the volume, for the labeling
reaction to be more efficient, and so that I will be able to decrease the salt
concentration by adding buffer A. I will be ready for the 2nd purification.
I add less MTSL here because I already label once. So I want to have 10
umole MTSL into each tube.
Make 100mM MTSL (in -20 freezer, blue box) spin labeling reagent (Dissolve
~ 26 mg of MTSSL in 1000 L absolute EtOH, : measure out MTSL in the
dark!
Add 100 L of MTSL reagent into each 50ml conical tube (1L growth, one
mutant)
React in the dark (use foil or put them in the drawer) at 37 degrees
Purification 2 (I skip this step for single mutant prep)
-
-
Add 10 or 15 mL of buffer A into the 5 mL reacted protein. So you will inject
15 or 20 mL of protein.
You need to dilute the sample with buffer A to dilute the salt, so that the
protein can bind to the column.
Sometimes, the protein goes out later, so there will be more salt in your
sample, so you will need to dilute more. I usually make sure the “initial” salt
concentration before injection is 100 or 150 mM LiCl. For instance, if the
protein is eluted with 400 mM LiCl, and that I have 5 mL of concentrated
protein, when I am adding 15 mL of buffer A, I dilute 4 times, so that my
final concentration of LiCl will be 100 mM before I inject on the column.
Purify using program “wash for fecA”
Gel 2 (I skip this step for single mutant prep)
Pool the sample together. The protein usually precipitates from fractions 22 to 28.
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Bradford Assay Protocol for membrane proteins
YOU NEED TO HAVE THE CONCENTRATIO OF THE WT AND THE DOUBLE MUTANT
***Important: wear gloves for entire procedure
- When we prepare our buffer A and B, we add 5 g of OG for 1 liter of buffer,
so we have 0.5% of OG (0.5 g of OG for 100 mL of buffer)
- When you read on the Coomassie documentation, they say the compatibility
limit is 0.5 %, so I think it is OK.
- BUT do not do a Bradford after you concentrate your protein. Because when
you concentrate the protein, you concentrate the detergent too! So you will
be higher than 0.5 %, and that will interfere with the Coomassie reagent.
1) Remove appropriate amount of Coomassie Reagent from cold room to
warm up to room temp.
Mix big bottle of Coomassie Reagent thoroughly upon removal from cold room as
solution may not be homogenous.
total volume of Reagent = (1.5 mL) x (# of samples, including standards)
2) Standards (BSA, bovine serum albumin)
Pre-diluted samples in cold room OR undiluted 2,000ug/mL ampules at RT
Typical set: 1000 ug/ml
750
500
250
125
50
25 <= if NOT pre-made (20uL of 125g/ml + 80uL of H2O)
0 (use water instead of using standard solution)
For Standard!! (Culture tubes: 12 x 75 mm works best)
50ul total volume of each standard
50ul total volume of buffer (containing detergent) 60%A + 40 %B
1.5ml of coomassie solution
3) Sample
50ul total volume of sample in detergent
50ul total volume of ddH2O
1.5ml of coomassie solution
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4) Getting UV-Vis ready
Open “Concentration” program
Click “Setup”
First Tab “Cary”
Wavelength 595 nm
Click “Samples/Std Averaging”
2
Second Tab “Standards”
Change units to mg/L
Standards
7
Type in the concentrations for each Standard (ie, 0, 25, 125, etc)
Click quadratic on “Fit Type”
Ignore Third and Fourth Tabs
Fifth Tab “Samples”
Number of samples
4 (for example)
Type in Sample Names
Because standards are duplicates, program expects duplicates for
samples. Simply use same sample. Read it twice.
Sixth Tab “Report”
Fill in name of operator.
Comment usually “Bradford Assay and date”
Deselect company logo.
Seventh Tab “Autostorage”
Click on “Storage on (Prompt at end)”
Click OK and computer program is ready to go!
5) Add 1.5ml of room temp Coomassie reagent to each standard and sample.
VERY IMPORTANT: Vortex gently to mix reagent and standard/sample.
6) Read samples
- Blank the instrument: Use 0ug/mL as the zero concentration and hit “Zero”
- Hit “Start”
- Click on the double arrow key to transfer all standards and samples to the
left column.
- Hit “OK”
- Insert each standard and sample as called for by the program.
- At the end, to add points to the graph, click on graph, trace settings, and
check standard calibration.
Do a Bradford assay, to estimate the protein concentration
Mix with WT, with a molar ratio of 3 to 1, or 6:1..
So, for one dialysis tubing, we need 0.75 mg WT and 0.25 mg of double labeled protein.
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Reconstitution into POPC vesicles
You need,
* 1X Dialysis buffer (4L beaker)  reconstitution buffer or EPR buffer
1)
2)
3)
4)
Dissolve HEPES 9.5 g and NaCl 30 g in ddH2O
Adjust the pH to 6.5
Fill up to 4L with ddH2O
Add 1mL of 1M NaN3 and 32 L of 0.125 M EDTA
Note: you can put maximum 4 samples in a 4L beaker.
You need to change the buffer 6 times to complete the dialysis, which
means you need a total of 24L of 1X dialysis buffer.
So, one easy way to avoid make buffer every time when you need it is to
make 10X dialysis buffer for storage. And whenever you need 1X buffer,
prepare 1X by diluting 10X buffer with water and adjusting pH!
-
Take OG (detergent) and POPC (lipid) from the -20 freezer, let them at RT.
Pool the pure protein fractions based on the gel in a 50 ml conical tube
-
(This is for one sample where you want 1 mg of total protein, either single or
double mutant. For single mutant, you do not need to dilute with WT. For double
mutant, you need to dilute in ratio 3WT:1 prot, or 6 WT:1 prot.
-
If you want to reconstitute many samples at the same time, you need to multiply
the amount of the lipid, OG, and buffer depending on the number of mutants and
then split 1ml into each mutant. I usually do 2 dialysis tubing, with 1 mg of protein
inside each dialysis tubing).
-
For double mutants:
-
We want a ratio of 3WT : 1 double mutant, corresponding to 0.75mg WT and 0.25
mg double mutant
-
We want a ratio of 1 protein :25 lipid
-
And a ratio of 1 lipid : 4 detergent
So finally, we will have 1 mg total prot / 25 mg lipid / 100 mg OG,
So if you want to do 2 dialysis tubing, you will need to double the amounts!!!
In a 50 mL conical tube
1) 25 mg POPC (lipid) + 1 mL 1X dialysis buffer
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2) Sonication for 5 min  cloudy solution
3) Add 100 mg of OG
4) Sonication for 5-10 min  clear solution (here, you can skip step 2 and you
can mix POPC and OG together and sonicate) but sometimes, can see that the
POPC is not well dissolved. Can see some flake.
5) Add 1 ml of buffer containing POPC+OG into the pooled protein sample (1
double mutant mixed with WT)
6) Fill it with buffer A up to 30 ml for dialysis tubing
7) Let it shake on the shaker on my bench for 40 min to make sure there is no
more flake  becomes clear
8) Put the sample into either dialysis tubing (in the cold room  you need 1
white closure, 2 red closures, and 1 buoy)
9) Use stirrer for dialysis
10) Change buffer 6 times for 3 days (for example, mon am, mon pm, tue am, tue
pm,wed am, and wed pm)  you should dialyze enough to get rid of all the
OG molecules!!
Sample Concentration
(pelleting down the large unilamellar vesicles containing proteins)
1. Centrifuge using ultracentrifuge
-
2. Airfuge
-
-
Shake dialysis tubing or cassette and take out sample slowly from it.
You have 2 dialysis tubing, containing 30 mL sample each. So you can split
into 3 ultracentrifuge tubes with 20 mL into each. You will need to
equilibrate the rotor.
Balance samples with dialysis buffer 1X
Spin down at 30 000 RPM for 30 min using the ultracentrifuge
Decant the supernatant and keep the pellet. You will have 3 pellets
Add 30 L of the dialysis buffer 1X to each pellet and resuspend. The final
volume will be around 200 uL into each ultracentrifuge tube.
Add 200 L of the resuspended sample into an airfuge tube
Spin it down at about 20 psi for 30 min
Decant the supernatant
Add more sample into the tube and spin it down again  after the final run,
take out the supernatant, and do it again (I usually keep the supernatant,
because there might be not enough sample for the last centrifugation. So I
can add some of the supernatant to equilibrate the airfuge tubes)
Take out supernatant  store the pellet in -20 freezer!
Now, you’re ready for EPR!
-
Depending on the size of the pellet, I can add 5 or 7 uL of dialysis buffer 1X
before doing EPR.
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Check the CW EPR spectra of the double mutant, and compare with the spectra of the 2
single mutants.
If you think both sites are labeled, you can try DEER.
23