CELLS
Tissue Culture Media for Hepa1a cells with Z and M AAT
H1A media
Minimum Essential Medium
Non-essential amino acids (100 x )
Waymouth MB 752/1 Medium
Fetal Bovine Serum (batch A)
10 x Penicillin/Streptomyocin
100 x Glutamine
300 ml
5 ml ( to 1 x)
100 ml
50 ml (10%)
5 ml (to 1 x)
5 ml (to 1 x)
#11095-080
#11140-050
#11220-035
(store)
(store)
(store)
500 ml
1 ml (0.1mg/ml)
#10131-035
500 ml
4 ml (0.4mg/ml)
#10687-010
G418 media (for MM15 cells – M AAT)
H1A media
G418 (geneticin)
(50mg/ml)
Hyg media (for Z-hg cells – Z AAT)
H1A media
Hygromycon B
(50mg/ml)
** Cat numbers for Gibco media / Invitrogen
New Protocol for Making Competent Cells
Solutions and Media
 2YT or LB media
 TSS (Transformation and Storage Solution for chemical transformation)
85% LB medium, 10% (w/v) PEG8K, 5% (v/v) DMSO, 50mM MgCl2,
pH6.5.
This solution can be autoclaved (or filter sterilized) and stored at 4oC for
2 weeks, or –20oC forever.
LB medium: 10g Tryptone, 5g Yeast extract, 10g NaCl, dissolve in 1L
H2O
Method
 Inoculate a colony or 200ul overnight culture to 20ml of 2YT or LB
medium. Shake the medium at 37oC to OD600 about 0.3-0.5.
 Chill the cell culture on ice for 20mins.
 Spin the cells down: 3000rpm, 10mins, 4oC
 Resuspend the cells in 2mls of ice-cold TSS solution.
The competent cells are ready.
* DNA transformation is the same as the method on the old protocol.
* Before plating, the transformed competent cells may need to be diluted at
least 10 times.
Autoinduction Small Scale Growth
Media:
60g Merck Overnight Express
10mL glycerol
in 1L water
Autoclave media, make only a small amount as it shouldn’t be used after 1 week storage
Add 2mL AI media to each well of a 24 well expression plate
Add appropriate antibiotics to wells
Add 50uL of overnight culture to each well
Grow for around 16hrs at 28C, shaking at 300rpm (small incubator in D119)
** These cells should be thick, and would ideally like to have OD600 at around 14
Freezable Competant Cells
Buffers
TFB1:
100mM RbCl (1.2g), 50mM MnCl2 (845mg), 30mM K acetate (294mg),
10mM CaCl2 (147mg), 15% glycerol (15ml), pH 5.8 (up to 100ml)
*** adjust pH with 0.2M acetic acid, pH drops fast to be careful
TFB2:
10mM MOPS (105mg), 10mM RbCl (60mg), 75mM CaCl2 (551mg),
15% glycerol (7.5ml), pH 6.8 (up to 50ml)
*** adjust pH with KOH

Pick a single colony and inoculate 10ml of 2YT media. Grow overnight at 37C

Add 1ml of overnight culture to 100ml 2YT media and shake at 37C until an
OD600 of 0.5 is reached

Cool the culture on ice for 5 min

Collect cells by centrifugation at low speed (10min, 3000 rpm)

Discard the supernatant carefully. Always keep cells on ice

Resuspend cells gently in cold TFB1 buffer (30ml for 100ml of culture) and keep
suspension on ice for 90min

Collect cells by centrifugation (10min, 3500 rmp)

Discard supernatant carefully, keeping cells on ice

Resuspend the cells carefully in 4 ml of ice-cold TFB2 buffer

Prepare aliquots of 100 ul in sterile tubes and freeze in dry ice-ethanol mix. Store
at -70C for up to 6 months
Checking growth rates of the proteins
Want to check whether the various cell lines are growing at different rates. Want to
check the total protein generated by the cells as they are growing.
-
Trypsinise cells from first plate so that a split ratio of 1/40 will be used. Need to
dilute a 60 mm plate to 5 ml, then add 65ul to each new 20mm well. Want the
cells to be able to grow for 5 days.
-
Add 65uL of cells to 2 ml of media in each 20mm well. Make up 5 dishes per cell
line. Swirl and make sure there can be uniform cell adherence to plate.
-
Keep some cells for NP40 lysis as a 0 time control point.
-
Each day, wash cells with PBS, then add NP40 lysis buffer and scrape cells of
plate (no TE treatment). Lyse cells, spin out nucleus etc. Freeze samples.
-
Monitor total protein using BCA assay in plate reader.
-
Resuspend cells in small volumes initially, as there would be fewer cells, then can
increase the volumes later.
-
When BCA has determined the protein concentration, convert mg/ml protein to
total protein (mg) taking into account what volume of lysate was used to
resuspend the cells in the first place.
-
Keep some media before ditching from the cells each day for a secreted assay
again.
Cell Counting

Treat cells with trypsin/EDTA and resuspend in media

Ensure that the resuspension is very vigorous in order to get single cell suspension

Mix 10 uL of cells with 10 uL of Tripan Blue on the side of a heamatocrit slide

Pipette 10 uL into the space between the slide and the square in the middle of the
slide

Count the cells in each of the four corners of the larger square, adding them
together to get a total for the cells

Only include cells that lie on the top and right hand lines so that cells are not
counted twice

Make sure the lens is on the 0.25 lens for counting on the microscope
To calculate the number of cells per mL:
N/4 x 2 x 104 = cells/ml
Where N = total of cells from all four corners
MOLECULAR BIOLOGY
Sequencing
The BigDye Terminator kit was used to sequence all plasmid DNA.
Reaction Mixture
Template
2.0l
T7 promoter (10M)
0.5l
BigDye Terminator (BDT)
5.0l
Sterile ddH2O
12.5l
The reaction mixture was subjected to PCR temperature cycling under the following
conditions.
Step
Temperature
Time
Repeats
Initial Denaturation
90 oC
1 minute
Denaturation
90 oC
1 minute
Annealing
50 oC
1 minute
Extension
60 oC
4 minutes
Final Extension
60 oC
4 minutes
1
Hold
4 oC
5 minutes
1
1
25
PCR product clean-up.
Following PCR, 2µl of 0.3M sodium acetate (pH 5.2) and 40l of ice cold 100% ethanol
was added to the PCR product and transferred to a 1.7ml eppendorph. The solution was
then votexed and chilled at –80oC for 20 minutes. The PCR product was then centrifuged
at 13,000rpm for 10 minutes and the supernatant removed and discarded. 100l of ice
cold 70% (v/v) ethanol was added and centrifuged at 13,000g for 5 minutes, the
supernatant was again removed and discarded. This step was repeated once to remove
any excess nucleotide bases and contaminating salts that could impede sequencing. The
remaining ethanol was evaporated by heating at 90oC for 5 minutes. The PCR product
was then sequenced at Micromon (Monash University, Australia) using an Applied
Biosystems 3730S Genetic Analyser. the automated system at Micromon Sequencing.
PROTEIN ANALYSIS
Silver Staining method
1.
Fix with fixing solution for 30 min.
2.
Washing with 30% ethanol 10-15min 3X
3.
Sensitize with Solution A for 1 min
4.
Wash 2x for 1 min with dH2O
5.
Stain with Solution B for 20 min
6.
Wash with dH2O once for 1min
7.
Develop the gel in 100ml of Solution C, watching for the appearance of bands.
8.
Stop development by adding 5ml acetic acid while shaking. NOTE: be careful of
the bubbling that occurs when you add the acid. Leave for about 2-3 min.
9.
Rinse gel in water.
Make up solutions fresh each time.
Fixing solution – 30% ethanol, 10% acetic acid, in 100ml water
Solution A – 37mg Na2S2O3.5H2O in 100ml water
Solution B – 0.2g AgNO3 (silver nitrate), 75l formaldehyde in 100ml dH2O
Solution C – 6g Na2CO3, 45l formaldehyde, small rock of Na2S2O3.5H2O (0.5mg) in
100ml water
Protocol for setting up AUC cells and samples
Sample preparation

Ideally samples are passed through a gel filtration column before being
taken for AUC to remove aggregates and unwanted material

Samples should be spun to remove any precipitated material before
checking OD280

Check OD280 of each sample before setting up cells. Need to ensure that
samples are between 0.3-1 ideally, however can get away with samples
0.1-0.3 as well

Use small cuvette (1mm, 120ul cell), buffer blank first with same buffer

Read spectra of sample to ensure OD is ok before commencing
Cell set up

Before adding sample, tighten the cell.

Place cell in the holder and push down, while tightening the holding jacket.
Gently lower the extension arm into the slots on the top of the cell. Insert
the torque arm into the top of the instrument, and turn clockwise firmly.
Keep turning until arm feels as stiff when the top reading is at 120.

Remove cell from device being careful not to allow the extension arm to
drop onto the cell.

Remove cell and place in Perspex holder with writing facing you and filling
holes on top. When handling cell, ensure the top is facing you. The top
has the writing “OUT” written on it.

Each cell contains 2 chambers, one will always contain buffer, the other
will always contain sample. Always have buffer on the left and sample on
the right.

When taking up sample into filling syringe, leave a small gap before buffer
it taken up, which will aid in expelling the full amount of sample later.

Inject 400 ul of buffer into left hand filling hole using specially adapted
syringe. Be careful not to introduce too many bubbles to the sample.

Inject 380 ul of sample into the right filling hole as above.

Check that the meniscus of the buffer is above that of the sample. If not,
the correct.

Carefully remove a small Teflon washer from the container (ensure there
is only one used) and place into each of the filling holes.

Insert a small screw into each filling port and tighten carefully with small
screw driver. Tighten simultaneously to ensure pressure is even. Tighten
to ‘finger tight’.
Placing cells in rotor

Remove rotor from its bag

Push all cells being used into the appropriate holes, with the screws facing
in, and the writing facing up.

Use a special tool to push cells to the bottom of the holder

Turn rotor onto its side, careful not to bang it on the table

Align cells using a twisting tool. Use the magnifying glass to make sure
that the hairline markers are in line with each other

Gently return the rotor to a horizontal position
Placing rotor in the instrument

Carry the rotor by its handle to the centrifuge.

Carefully place the rotor on the spindle, making sure there is no
movement of the spindle.

Gently spin the rotor in a clockwise direction to ensure it is sitting correctly.

Remove the absorbance detector from its box and carry to the instrument.

Insert the arm into the rotor with the pins aligned.

Carefully hold the arm and push down on the red locking nut in order to
lock in place. Be exceptionally careful not to knock the rotor while doing
this.

Turn on the instrument and activate the vacuum.
Starting a run

Ensure instrument is turned on

Open Proteomics package on computer to control the instrument

Initially need to run a radial scan and calibration
Protocol for ELISA assay for Protein A
Buffers/Reagents
Coating buffer
PBS
(or packet)
PBS/Tween 20 solution
Blocking solution
0.8g Na2CO3, 1.46g NaHCO3 in 500 ml H2O, pH 9.0
0.26mM KH2PO4, 2.7mM KCl, 0.08% w/v NaCl, pH 8.2
Antibody
conjugate (A3687)
Substrate
(AB0100/AB0200)
Stop solution
Sigma IgG Anti-rabbit (Goat) alkaline phosphatase
0.1% v/v in PBS
5% w/v milk powder in PBS/Tween solution
Red Microwell subtrate for alk phos Sigma
Sigma solution (A5852)
Procedure
Add 150uL of coating buffer to each well to be used
Add 50uL of sample to the first well, then make serial dilutions by removing 50uL and
adding to the following well, in sequence. Remove 50uL from the final well and discard.
Incubate plate at RT for 2hrs, or 4C overnight. Expel solution rapidly.
Wash plate by filling used wells with PBS/Tween solution, then expelling rapidly. Bang
on paper towel to remove excess solution. Perform 2 times
Block remaining sites with blocking solution by adding 200uL to remaining wells and
incubating at RT for 1hr.
Wash plate by filling used wells with PBS/Tween solution, then expelling rapidly. Bang
on paper towel to remove excess solution. Perform 2 times.
To bind antibody, dilute the IgG 1:30,000 in PBS/Tween solution, then add 200uL to
each well. Incubate at RT for 2 hrs.
Wash plate by filling used wells with PBS/Tween solution, then expelling rapidly. Bang
on paper towel to remove excess solution. Perform 4 times.
Bring both solutions to RT. Mix equal volumes of the two subtrate solutions, then add
200uL to each well incubated at RT for 30min. Add 50uL of stop solution to each well to
end reaction. Read in a plate reader, using a wavelength of 490nm (can use between 45055-nm).
kass protocol for ACT proteins
Solutions:
Activity Assay Buffer –
100mM Tris
50mM NaCl
10mM CaCl2
0.025% PEG 8000
pH 7.4
(made up to 10x stock and stored in 10ml aliquots)
Enzyme – chymotrypsin, 80uM
Substrate – pNA , 20mM
Dilutions:
pNA: dilute to 2mM, need 20uL per well
chymo: double dilution – 1uL up to 1ml ([chymo] = 0.08uM)
10uL of this up to 4ml ([chymo] = 0.0002uM
ACT: dilute to 0.05uM
Set up:
1.
2.
3.
4.
Add pNA to wells
Add buffer to wells
Add ACT to wells
Add chymotrypsin with multichannel pipette
Fibril Protocols
Setting up samples:



Determine stock concentration of protein by a combination of Abs280 and BCA
assay. Make sure both correlate if not exact take the average of both. It is
important that the stock concentration is correct as all other calculations and
concentrations depend on it.
The fibril reactions are done in the presence of both 1mM DTT and 2mM PMSF.
A stock concentration of 180mM PMSF should be made up. Make sure you make
up each week as PMSF will breakdown overtime and become ineffective.





DTT is usually made up to 1M and is stable in the freezer.
All fibril reactions should be done in PBS/10% glycerol (pH 7.4).
If adding PBS to fibril reactions add stock with 10% glycerol!
Samples are placed in incubator and time on front recorded in book.
Gels should be run of most assays to double check that no protein cleavage is
occurring.
Taking Readings: For a reaction concentration of 65uM:




I find it works well for most measurement types to take the samples, freeze them
at -80C and do the all the readings at one time.
7µl for ThioT (Add 490µl of 25µM ThioT to 7µl sample and measure emission).
10µl for Slot Blots (See protocol or Austin).
3µl for gels and western blots (add 20µl LD, boil and load 7µl into each gel lane).
ThioT Readings:
ThioT is made to 25uM in filtered PBS (no glycerol)
1. Take 7ul sample and place in tube.
2. Go to read application in FLwinlab. Configure to excitation wavelength 440nm,
emission wavelength 485nm, excitation slit width 5nm and emission width 10nm.
3. Wash a 500ul fluorescence cuvette with water, dry, and place in holder.
4. Add 490ul of ThioT in cuvette. Take reading, this will be your blank reading.
5. Add 490ul of ThioT to tube with protein inside and use a glass transfer pipette to
add sample into empty cuvette.
6. Take reading (write down the second reading or from when it has stabilized).
Suggested plan of attack for the next two months
1. Prep some protein; 5-10mg should be enough to make a good start.
- Store in 55ul elution’s at 4mg/ml.
- Have gels of each stage and traces from computer, these will be used in
your thesis. Scan gels in early so it is of no stress later.
- If this form of the prep causes you too much trouble, you may have to
revert to the HISQ50 prep ie, no GST Michelle can help you with this
prep.
2. Set-up protein at 65uM and follow with ThioT, Slot Blot and a SDS gel.
- The gel will make sure this is no protein cleavage.
- Do a western of the gel as it will be useful for your thesis.
- Repeat 2 more time or until reproducible.
- Get EM pics of the reaction as they look pretty in your thesis.
3. Set-up same concentration as standard and add additives to separate reaction
tubes. Follow with slot blot and compare to standards to gauge any effects
occurring.
-Further characterize the additives having an effect by EM/gels and anything else
Steve suggests.
Ways of observing ANS binding from Mike
1 – Titrate in protein into a fixed ANS concentration, and mop up all the ANS. This will
result in the maximum ANS signal possible when that amount of ANS is all bound. Must
get to a very high level of protein concentration then.
- Determine what the maximal fluorescence change is for that amount of ANS.
- Titrate fixed protein with increasing amounts of ANS and observe the increase in
fluorescence. With increasing ANS the curve will appear to plateau but it will curve up a
bit. As a control titrate ANS into buffer with no protein present and observe the same
curve up phase. Subtract control from the protein curve and look at the maximum
fluorescence
seen.
fluorescence
fluorescence
Max fluorescence with fixed ANS
F
Fx N
Protein
ANS
the change in fluorescence seen for increasing ANS present will represent the number of
sites present ie. If maximum fluorescence is 20 for increasing protein, and the maximum
is 40 for the ANS titrated sample, then there are 2 binding sites (N)
2 – The other way is a little more complicated, but should be better if there are changes in
the affinity etc of the interaction between the mutant and wild type proteins.
Titrate increasing amounts of protein into a solution with fixed ANS concentration.
Make sure that there will be enough protein to well and truly mop up all the ANS present.
Need to take into account the kD of ANS, which is probably pretty high, i.e. will need to
have the concentration of the protein present in the assay at least as high as the kD times
the ANS concentration. Need an excess.
- determine the maximum change in fluorescence for this (as in the first graph of
the previous test)
- calculations then need to be made: calculate Q: fractional saturation which is the
fluorescence at each point divided by the absolute maximum fluorescence
possible
- divide the protein concentration by Q for each point
- determine 1/1-Q for each point
Plot these on a graph (Klotz plot):
1/1-Q
1/kD
1
n/Q
No. sites x molarity of protein
Using this plot the numbers will never be below 1. the data should cross the x axis in the
positive range.
TUG Gel Protocol
Gels for ACT proteins: pH 7.5, 7.5% acrylamide, 5M urea
Buffers
5 x TUG buffer
215 mM Imidazole
175 mM HEPES
14.64g
41.7g
up to 1 L (should be pH 7.5)
Gel Solution A – low urea
5 x Buffer
Acrylamide
dH2O up to 10 ml
2 ml
2.5 ml (final = 7.5%)
Gel Solution B – high urea
5 x Buffer
Acrylamide
Urea (solid)
dH2O up to 10 ml
2 ml
2.5 ml (final = 7.5%)
3 g (final = 5M)
Protocol
 Set up gradient pourer over a stirrer, attaching the channel to a P1 pump
 Have flea in the channel closest to the pump
 Add Solution A to the furthest channel from the pump and prime the line
 Add Solution B to the channel closest to the pump
 Add 4 µL of APS and 1.5 µL of TEMED to each channel, then turn on pump
(slowly, about 4) and open line between channels
 Pour into vertical plates
 Allow to set
To Run Gels




Dilute 5x buffer to 1x (160 ml in 800 ml)
Use continuous buffer system (same in both chambers)
Run 1 gel at 15 mA or 2 gels at 25 mA
For ACT proteins, gels run approximately 2½ hrs
Setting up gel plates

Apply a thin coat of Vaseline to the U spacer to prevent leakage of gel



Set up plates as per Fig 1, making sure to leave an overhang of the spacer for
removal with tweezers after gel has set.
Add bulldog clips to gel set up to seal. Use a large clip for the bottom and fold the
arms up to the gel to provide a flat base to sit the apparatus on. Careful to place
clips onto spacers so as not to crack plates.
Pump gel from the gradient pourer 8M first to settle at bottom.
Fig 1
Plates
Gel Solution
0M
GEL
8M
Overhang
U spacer



Once gel has set remove bulldog clips and use tweezers to gently remove the U
spacer.
Rotate the gel 90° anti-clockwise to have left to right 0-8M gradient.
Add spacers as per Figure 2, being sure to have spacer right to the edge of the gel
plates (this will prevent breakage of plates when put into the gel holder)
Spacers
Fig 2
Top
GEL
Bottom
0M

8M
Slide gel into holder (Fig 3) with slight overhang at the bottom and finger tighten
screws to hold plate (careful not to tighten too much as to break plates)
Fig 3
Top
SCREWS
GEL
SCREWS

Fill the space between the gel plates at the bottom of the gel with 1x TUG gel
buffer making sure to have no trapped air to allow proper current flow.
 Put gel into tank half filled with 1x TUG gel buffer and ensure not air is trapped
between the bottom of gels
Load protein evenly on top of the gel and run as