COS-7 cells were transfected, see 2:6 i, with 20 pg GFP or 20 |ig pervin- FLAG/headless pervin-FLAG and left for 1, 2 or 3 days at 37°C. The dishes were placed on ice, the media removed and the cells washed twice with ice cold PBS. 400 pi of IX Lysis Buffer (1 mM MgCL, 75 mM KCl, 2.5 mM EGTA pH 8, 1
mM KPO4, 5 mM |3-glycerol phosphate 0,1 mM DTT, 2.5 mM ATP, 1% Triton
X-100 and protease inhibitor cocktail (Roche)) was added to each dish, left for a few minutes and then the cells scraped from the dish and transferred to an microfuge tube. Then spun at 5, 000 G, 4°C for 20 minutes. The Triton X-100 soluble supernatant representing the cytoplasm and non-cytoskeletal associated elements was removed and mixed with an equal volume o f 2 X sample buffer. The pellet representing Triton X-100 insoluble cytoskeletal elements was resuspended in 250 pi of 2x SDS PAGE sample buffer (100 mM TRIZMA Base® pH 6.8, 4 % SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM DTT). Both
the pellet and the supernatant were denatured for 3 -5 minutes at 100°C and stored at -2 0 X until analysed by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), see 2:8
2:7 Hi: Determination o f protein concentration using the Bradford Assay
The Bradford assay quantitates by optical density the binding of Coomassie brilliant blue to a protein. Under acidic conditions the dye is in a red, doubly protonated form. After binding to protein the dye looses the protons and turns blue. This is compared to the binding o f Coomassie brilliant blue to different amounts o f a standard protein, eg bovine serum albumin. This protocol quantifies 1 to 1 0 pg protein using a standard curve.
2:7 iv: Creating a standard curve
A protein stock solution of 0.5 pg/pl was made by mixing 50 pi 10 mg/ml BSA (Fraction V), (made in a tube previously coated with BSA) with 950 pi distilled water. To create the samples required for the standard curve 0,5, 10, 20, 30 and 40 pi of 0.5 pl/pg BSA stock solution were added to a 1 ml of Bradford Reagent diluted 1:4 with distilled water. This gave final BSA concentrations of 0, 2.5, 5, 10, 15 and 20 pg respectively. The samples were vortexed and their OD reading taken at 595 nm. The readings were then plotted as OD versus BSA concentration. 1 0 pi o f each sample with unknown protein concentration was
mixed with 1 ml of Bradford Reagent diluted 1:4 with distilled water and vortexed. Each sample’s OD reading was then taken as above. These results were then used to read off approximate protein concentrations using the standard curve.
Using this information it was decided to run between 30-100 pg o f protein/lane via SDS-PAGE, see 2:8.
2:7 v: Immunoprécipitation o f COS-7 cell lysates
COS-7 cells were transfected , see 2:6 i and left for 72 hours. The cells were rinsed 3 times with PBS phosphate buffered saline and solubilized with 1 ml of Lysis Buffer (50 mM TRIZMA Base® pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 % Triton X-100, ImM Na pyrophosphate, 1 mM Na orthovanidate, 0.5M NaF and protease inhibitor cocktail (Roche) pH 7.4). The cells were left on ice for 30 minutes and the dishes gently rocked occasionally. Insoluble material was removed by centrifuging the cell lysates at 17,000 G for 30 minutes at 4°C. 13 pl/ml of lysate of anti-p-actin monoclonal antibody or 5 pl/ml o f lysate anti FLAG M2 monoclonal antibody were added to the cell lysate supernatants and the
mixture incubated overnight at 4°C. 200 of Protein A sepharose CL-4B (Amersham Pharmacia) or 40 pi of Protein G Plus-agarose (Santa Cruz) was subsequently added and the lysate incubated for a further hour. The lysate was spun at 17,000 G for 5 minutes at 4“C and the pellet washed 3 times with Lysis Buffer. The pellet was resuspended in 50 pi o f sample buffer and boiled for 5 minutes prior to SDS-PAGE, see 2:8.
2:8 Electrophoresis of protein samples
The protein samples were separated via SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) through a 6% stacking gel which is poured on top
of a polymerised 10% separating gel. 4-10 pi of Rainbow Marker (Amersham Pharmacia Biotech) was used and the gel run at 7 mA and 250 V over night or
150 V for 8 hours.
2:8 i: Protein to membrane transfer
The electrophoresed proteins were transferred to Hybond-C extra membrane via electrophoretic elution. A sandwich was made consisting of a piece o f transfer membrane and the gel between adsorbant filter paper. This was submerged into a transfer tank filled with transfer buffer (39 mM glycine, 48mM TRIZMA Base®, 0.037% SDS, 20% methanol) with the membrane closest to the positive electrode (red). Transfer occurred between 5 hours to over night at around 13 volts, maximum current
2:8 ii: Detection o f proteins using ECL ™
The primary antibodies, anti-P-actin monoclonal antibody and anti-FLAG M2 monoclonal antibody were detected using highly sensitive ECL™ reagents (Amersham Pharmacia Biotech). A secondary horseradish peroxidase antibody
(Amersham Pharmaica Biotech) is capable of catalysing the oxidation of luminol in alkaline conditions, potentiated with phenol. Immediately after oxidation luminol is in an excited state and will decay back to ground state via a light emitting pathway (emission at 428 nm). The protocol laid out in the ECL manual was followed. This method will detect 1 pg of antigen or less on Hybond-ECL, with high resolution.
The membrane was removed from the transfer tank and the marker bands marked onto the membrane. Non-specific binding sites on the membrane were blocked for 45 minutes using 100 ml of blocking solution (5% powdered milk and 0.1% Triton X-100 in PBS). The membrane was heat sealed into a small plastic bag containing 6 ml of Blocking solution or 0.1% Triton X-100 in PBS and 10-40
pi of 1 pg/pl anti mouse FLAG monoclonal antibody or 20 pi o f anti-P actin antibody. This was left shaking at room temperature for 1 hour or at 4°C over night. The membrane was then washed, shaking, for 10 minutes 3 times in 150 ml o f Wash buffer (0.1% Triton X-100 in PBS). 3 pi o f anti mouse-HRP (Amersham Pharmacia Biotech) and 4.5 ml of Blocking buffer (1:1500 dilution) was added to the membrane in a heat sealed bag and left for 1 - 2 hours at room
temperature. The membrane was washed 3 times as above. Wearing gloves 1 ml each o f Solution 1 and 2 (from the kit) were mixed on a hard-wearing piece of plastic. The excess Wash buffer was drained from the membrane and it was placed face down on top of the 2 ECL ™ solutions and incubated for 1 minute at room temperature. After draining off excess solution the membrane was wrapped in Saran wrap, smoothing out any bubbles. The membrane was then exposed to X- omat film for 30s to a few minutes and the film developed by washing firstly in
developer and then in fixer (Champion Photochemistry Int Ltd; Devalex Developer, Devalex Developer Starter and Fixaplus).
2:9 DRG culture and transfection 2:9 i: DRG culture
DRG were dissected from new bom rat pups and placed immediately into Dulbecco's MEM with L-glutamine, 0.5 U/ml penicillin and 0.5 pg/ml streptomycin and washed in PBS minus Ca^'^/Mg The DRG were then left for 30 minutes to 1 hour in a final concentration of 0.125% collagenase XI at 37°C. The cells were triturated gently 3 times with a 19 gauge needle and then up to 10 times with a 21 gauge needle to break up the ganglia and release single cells. The media containing the cell suspension was gently laid over 70 pm nylon filter (Falcon) and then washed at 89 G for 5 minutes to pellet the single cell suspension. The cells were resuspended in Dulbecco's MEM with L-glutamine containing 10% foetal calf serum, 0.5 U/ml penicillin and 0.5 pg/ml streptomycin and 50 ng/ml NGF (Promega) and cultured in the centre o f 0.05 mg/ml poly lysine coated glass cover slips at 37°C.
2:9 ii: DRG transfection
DNA was transfected into DRG neurons using the PDS-1000/He Biolistic particle delivery system (Biorad) according to the manufacturer’s instructions. All solutions where possible underwent 0.22 pm filtration. This method uses pressurized helium to introduce DNA-coated sub-cellular microcarriers into cells over a range of velocities.
rupture disk, and partial vacuum to propel a macrocarrier sheet loaded with millions o f microscopic tungsten or gold microcarriers toward target cells at high velocity. The microcarriers are coated with DNA or other biological material for transformation. The macrocarrier is halted after a short distance by a stopping screen. The DNA-coated microcarriers continue traveling toward the target to penetrate and transform the cells. The launch velocity o f microcarriers for each bombardment is dependent upon the helium pressure (rupture disk selection), the amount of vacuum in the bombardment chamber, the distance from the rupture disk to the macrocarrier (Fig 2.1b: A), the macrocarrier travel distance to the stopping screen (Fig 2.1b: B), and the distance between the stopping screen and target cells (Fig 2.1b: C), see Fig 2.1.
2:9 Hi: Preparation o f gold particles
The plastic macrocarriers were inserted into metal holders and autoclaved along with three stop screens per individual wrap. For the bombardment o f six dishes, 3 mg o f 1 . 6 pm gold particles were washed by vortexing vigorously with 1 ml of
sterile 70% EtOH for 4-5 minutes. The particles were then pelleted by spinning for 30 s at 15, 000 G and the supernatant removed. The pellets were washed and pelleted as above with 1 ml of 1 0 0% ethanol and the particle pellet was then
vortexed for 1 minute 3 times with 1 ml of sterile water and pelleted by a brief 30s spin. After the final spin all of the supernatant was removed and 50 pi o f sterile 50% glycerol was used to resuspend the pellet. This will give a concentration of 60 mg/ml gold particles. The following reagents were added to this tube in the order that they are listed below and vortexed continuously for 15 minutes.
a
b
B e fo re
Gas Acceleration Tube
Rupture Disk Macrocarrier DNA-coated Microcarrier Stopping Screen Target Cells A fter
Fig 2.1: T h e Biolistic PDS-lOOO/He P a rtic le d e liv e r y s y s tem .
A: T h e in stru m en t p re pare d fo r b o m b a r d m e n t o f eells in a petri dish w ith in the chamber. T h e a r ro w po in ts to the part o f the in stru m e n t sh o w n in detail in B. B: H igh p re ssu re h eliu m , re le a se d by the ru p tu re disk in a partial v a c u u m propel Is a m a e r o e a r r ie r shee t loaded w ith D N A co v e red gold m ic ro e a rrie rs t o w a r d s the eells at h igh velocity. T h e m a e ro e a rrie r is halted afte r a sh o rt d ista n c e by a stop screen w h ilst the g old m icro e arrie rs travel th ro u g h the stop screen to p e n e trate the eells below. D ista n c e s A, B and C are v a ria b le and alo n g w ith the helium p re ssu re and the a m o u n t o f v a c u u m in the c h a m b e r control the launch v e locity o f m ic ro e a rrie rs for ea ch b o m b a rd m e n t.
5 )Lil of DNA or a mix of DNA (total DNA 5 ^ig) 50 \i\ of2.5M C aC l2
50 fil of 0.1 M spermidine (base free, tissue culture grade)
The gold particles were allowed to settle by leaving the tube standing for 3-4 minutes and the supernatant was then removed. 140 pi of 70 % ethanol was added and the particles vortexed for 1 min and allowed to settle for 3-4 minutes. This process was repeated with 140 pi of 100% Ethanol and all o f the supematent removed. The particles were finally resuspended in 60 pi o f 100% ethanol. 10 pi of suspended gold was smeared onto the centre of the plastic in each maeroearrier placed in a petri dishes containing silica, then dried inside a tissue culture hood. They must be used immediately. A PDS-1000/He Biolistic particle delivery system (Biorad) was used to biolistically transfect the cells at 500 psi helium output pressure and a vacuum of 6-8 Hg inches according to manufacturers instructions. After transfection, fresh media was added to the DRG cultures which were analysed 24 and 48 hours later.
2:9 Hi: Measuring neurite outgrowth in transfected DRG neurons
The cells were fixed using 4% paraformaldehyde for 10 minutes and then images were captured using the FITC/TRITC filter of a Leica TCS NT confocal microscope and saved as TIFF files using Adobe Photoshop. The images were then analysed by computerised image analysis with Kontron KS400 2 Image Analysis Software (Imaging Analysis), using a macro (developed by Dr Christopher Thrasivoulou Royal Free Hospital UK). This involved converting the image into a binary image where the neurites and cell body are white and the
background is black. The program masks the cell body and measures the total length of the remaining white neurites in pixels. Pixel length was calibrated so that the neurite length could be converted into pm. All analyses were performed blind.