sulfate; Invitrogen) from 200 to 800 µg/ml or puromycin (from 0.2 to 0.6 µg/ml) or both (about 2 to 3 weeks). Drug resistant clones were picked and expanded in growth medium containing genticin (800 µg/ml) or puromycin (0.6 µg/µl) or both. Clones were screened for the expression of the appropriate protein by Western blotting or by confocal microscopy of the cells.
HT1080 cells stably expressing galactose-6-sulfatase (kindly gifted by Transkary-otic Therapies Inc, Cambridge, MA) were transfected with pSB-FGE-His6 (Mari-appan M., unpublished data) or pSB-∆34-68FGE-His6 plus puromycin resistance vector (10:1 ratio) using Lipofectamine 2000. Stable clones were selected as described above.
2.4.2.2 Transient transfection
Transient transfection was performed with Lipofectamine 2000 following the proto-col recommended by Invitrogen. Typically, 2µg of plasmid DNA were used per 3-cm dish. After 6 hr, the medium was replaced either with normal medium or medium containing doxycyline (BD Biosciences), and 24 hr later, the cells were harvested for further analysis by Western blot.
2.5 Biochemical Methods
2.5.1 Analysis of Protein
2.5.1.1 Protein estimation by BIORAD reagent Bovine Serum Albumin (BSA) stock solution 1 mg/ml Concentration range : 2 -16µg/µl
A standard curve was made using BSA in the range of 2 -16µg. 10µl of the sample was used for the protein estimation. The sample volume was made up to 800 µl with water. 200µl of the BIORAD reagent was added to each samples, vortexed and incubated for 3 - 5 min at room temperature, 200 µl from each tube was pipetted into ELISA strips and optical density was measured at 595 nm in the ELISA reader.
2.5.1.2 Solubilization of proteins 2 x Laemmli buffer
125 mM Tris-Cl, pH 6.8 4% SDS
40 Chapter2. Materials and Methods 0.004%Bromophenol blue
20% (w/v) Glycerol 10%β-mercaptoethanol
Protein samples after speed vac were resuspended in 1x Laemmli buffer. The resuspended proteins were boiled at 95◦C for 5 min, cooled on ice for 1 min, centrifuged at 13,000 rpm for 2 min and resolved by SDS-PAGE.
2.5.2 SDS-Polyacrylamide Gel Electrophoresis
The elecrophoretic separation of the proteins was performed through high-tris discontinual SDS polyacrylamide gel electrophoresis. A system with vertical oriented glass plates (16 x 16 cm; 1 mm spacer) was used. The gels were prepared as follows: first the resolving gel (12.5-17.5% acrylamide) was put between glass plates and covered with a layer of iso-propanol or butanol. After polymerisation of the gel was completed, butanol was removed and the space between glass plates was dried with Watmann paper. Then the concentrating gel was put on top of the polymerised resolving gel and the sample combs were introduced immediately.
After approximately 15 min polymerisation was completed and the sample combs were removed. The glass plates with the gel between them were fixed inside the electrophoresis chamber and covered with electrophoresis buffer. Protein samples were mixed with Laemmli loading buffer 1:1, denatured by 95◦C and centrifuged with 14,000 rpm. The supernatant was introduced into the stacking gel pockets.
Electrophoresis was performed for 2-3 hours with the constant current of 35 mA.
Electrophoresis buffer:
SDS (w/v) – 10g/10 litre
Glycine (w/v)– 144.27g/10 litre Tris (w/v) – 60.53g/10 litre
20% Ammoniumperoxisulfate (APS) in ddH2O
Ingredients Stacking gel(5%) Resolving gel(15%) 30% Acrylamide solution 0.4 ml 8.75 ml
1% Bisacrylamide solution 0.275 ml 2.74 ml
ddH2O 1.175 ml 1.4 ml
0.5 M Tris-HCl, pH 6.8 0.625 ml
1.5 M Tris-HCl, pH 8.8 4.3 ml
(10%) SDS 25µl 175µl
TEMED 2.5µl 14.58µl
(20%) APS 10µl 58.33µl
2.5. Biochemical Methods 41
2.5.3 Detection of proteins in polyacrylamide gels
2.5.3.1 Staining with Roti-Blue colloidal Coomassie
Colloidal Coomassie staining is one of the most sensitive staining protocols. Due to the colloidal properties of the dye, it binds with high specificity to proteins and only minimal to the gel matrix. This allows visualisation of proteins separated by SDS-PAGE with sensitivity as high as 30 ng of protein. Roti-Blue colloidal Coomassie was purchased from Roth and staining was performed according to the protocol as follows. Immediately after completion of electrophoresis, gels were incubated in the fixing solution for 60 min, shaking. The staining solution was applied on to the gels for 60 min. In special cases the gels were stained for several days, which improved the sensitivity of the staining. After staining, gels were incubated in the washing solution for 5-10 min and then kept in the stabilising solution or prepared for drying in the drying solution.
Fixing solution: 20% (v/v) methanol
8.5% (v/v) o-phosphoric acid Staining solution: 20% (v/v) methanol
20% (v/v) 5x concentrate Roti-Blue colloidal Coomassie Washing solution: 25% (v/v) methanol
Stabilising solution: 20% (w/v) ammonium sulphate Drying solution: 10% (v/v) glycerol
20% (v/v) ethanol
2.5.3.2 Staining with silver
Staining of proteins separated on SDS-gel with silver allows quick and effective visualization of even small amounts of protein. After completion of electrophoresis gels were incubated with shaking in fixing solution for 1 hour. After that gels were washed 2 times for 20 min in 30% ethanol and equilibrated for 20 min in water. Equilibrated gels were washed for 1 min in 0.03% Na2S2O3, washed with water and incubated in staining solution for 20 min at 4oC. Then the gels were washed in water twice for 30 sec and changed into a new chamber. The gels were developed in the developing solution until desired intensity of the bands is obtained and developing is stopped by 5% acetic acid. Gels were kept in 1% acetic acid or washed with water to prepare them for drying.
42 Chapter2. Materials and Methods Fixing solution: 10% (v/v) acetic acid
40% (v/v) ethanol Staining solution: 0.2% (v/v) Silver nitrate
0.008% (v/v) formic aldehyde Developing solution: 3% (w/v) sodiumbicarbonate
0.018% (v/v) formic aldehyde
2.5.4 Western blot (semi-dry)
Cathode buffer 40 mM aminocaproic acid, 20 mM Tris-Cl, 20% (v/v) methanol, pH 9.0 (pH was adjusted with free Tris base before addition of methanol) Anode buffer 75 mM Tris-Cl, 20% (v/v) methanol, pH 7.4 Phosphate-buffered saline (PBS) 140 mM NaCl, 2.7 mM KCl,
10mM-Na2HPO4, 1.8 mM KH2PO2, pH 7.3
PBST 0.05% (v/v) Tween-20 in PBS
Blocking buffer 5% milk powder in PBST
Glycine stripping solution 0.2 M glycine pH 2.8, 0.5 M NaCl SDS stripping solution 16 mM Tris-Cl pH 6.8, 2% SDS,
0.1 M -mercaptoethanol
Six pieces of 3 mm Watmann paper and one piece of nitrocellulose membrane were cut to the size of the SDS gel. Gel and membrane were equilibrated for 5-15 min in cathode buffer. The blot was assembled without air bubbles according to the following scheme:
cathode (-)↓
3x paper in cathode buffer GEL
membrane
3x paper in anode buffer anode (+)
For transfer, the current was set to 1 mA/cm2gel size for 45-60 min. The membrane was then briefly washed with PBST and incubated in blocking buffer for one hour at room temperature. Decoration with the primary antibody diluted in blocking buffer occurred overnight at 4◦C. After three 30-45 min washes with PBST, the membrane was incubated with horse radish peroxidase (HRP)-coupled secondary antibody,
2.5. Biochemical Methods 43 diluted 1:10,000 in blocking buffer, for one hour at room temperature. The blot was washed as described above. The blot was incubated with chemiluminescence substrate solution and visualized by Intelligent Dark Box II Camera.
2.5.4.1 Stripping of nitrocellulose membranes
To strip the bound antibodies wash the blot in 0.1 M NaOH for 20 min and wash thoroughly with 1X PBS for 5 min. Repeat washing for five times. Incubate the blot in 5% blocking solution for 30 min and probe with the desired antibodies.
2.5.5 Detection of radioactively labeled polypeptides
For detection of radioactively labeled polypeptides polyacrylamide gels were fixed in destaining solution for 15 min and then washed with water for 30 min. Then the gels were dried between cellophane layers in the gel-dryer for 2 hours. For visualization of the incorporated radioactive label the gels were exposed on to the Imaging plate (Fuji) overnight or longer period at RT. The image on the plate was analysed by the program Image reader (Fuji-Film, Vers. 1.4 E) with the help of the BAS 1000 phosphoimager (Ray-test).
2.5.6 Identification of proteins by Matrix Assisted Laser Desorption Ionisation/Time-of-Flight (MALDI-TOF) Mass Spectrometry
2.5.6.1 In gel digestion of proteins separated by SDS-PAGE
The stained bands were excised from the gel with a scalpel and transferred into a PCR cup. The excised bands could have been kept at 4oC for several days or frozen at -20oC. The analysed bands were cut into approximately 1x1mm pieces and the staining was washed away by the following procedure.
Washing Added 100µl of 25 mM NH4HCO3/water;
Incubated at 37oC for 15 min, shaking;
Supernatant was discarded.
Added 100µl of 50% acetonitrile, 25 mM NH4HCO3/water;
Incubated at 37oC for 30 min, shaking;
Supernatant was discarded.
Repeat the above step
Added 100µl of 100% acetonitrile Incubated at 37oC for 15 min, shaking;
Supernatant was discarded.
44 Chapter2. Materials and Methods
Drying Kept the cup open at RT for 5 min
Reduction Added 10µl of 10 mM NH4HCO3/water to cover gel pieces; Kept at 56oC for 1 hr;
Chilled on ice; Supernatant was discarded.
Carbamidomethylation Added 10µl of 25 mM IAAD, 25 mM
NH4HCO3/water to cover gel pieces; Incubated at RT in the dark for 30 min;
Supernatant was discarded. Added 10µl of 10 mM DTT, 25 mM NH4HCO3/water to cover gel pieces;
Incubated at 37oC for 10 min, shaking;
Supernatant was discarded.
Added 100µl of 50% acetonitrile, 25 mM Incubated at 37oC for 10 min, shaking;
Supernatant was discarded.
Drying Kept the cup open at RT for 5 min
Protein digestion Added 120 ng trypsin in 6µl solution (icecold);
to prepare the trypsin solution added 60µl 25 mM NH4HCO3/water to an aliquot of 2.5µl trypsin stock solution (0.5µg/µl 0.01% TFA/water); used trypsin from Promega: V5111, Seq. Grade Modified;
kept on ice for 15 min to swell the gel; if necessary add 25 mM NH4HCO3/water to cover the gel pieces;
Incubated at 37oC for 2 hour or overnight.
Peptide extraction Transferred supernatant to a new PCR cup; covered the gel pieces with 20µl 1% TFA/water; Incubated at 37oC for 10 min, shaking; transfer the supernatant to the new PCR cup.
Drying Dried pool of extracted peptides completely in a vacuum concentrator (Speed Vac)
Resolving peptides Added 10µlof 0.1% TFA/water (for MALDI-TOF-MS); used peptide solution for further Zip Tip purification or directly for mass spectrometry or stored it in a freezer.
2.5.6.2 ZipTip purification of the tryptic peptides before analysis by MALDI-TOF
Purification of the tryptic peptides with ZipTip is used to remove salts and other contaminants which could disturb the following mass spectrometry analysis. Before purification the ZipTip was equilibrated with 0.1% TFA/70% acetonitrile by pipet-ting the solution in and out of the tip several times with following washing with 0.1% TFA. Binding of the peptides to ZipTip matrix was performed by pipetting the solution through the tip for 5 times. The unbound components were washed away with 0.1% TFA. The peptides were eluted with 0.1%TFA/70% acetonitrile solution.
The eluate was used for MALDI-TOF mass spectrometry analysis.
2.5.6.3 Sample/matrix preparation for MALDI-MS (drying droplet method) For preparation of the matrix 1000µl of 50% acetonitrile/0.1% TFA was added to 5 mg ofα-cyano-4-hydroxycinnamic acid with following shaking for 1 min at RT and
2.5. Biochemical Methods 45 ultrasound sonification for 5 min. The solution was centrifuged in an eppendorf centrifuge at 13,000 rpm for 5 min. The supernatant was used for co-crystallization with the sample. For best results this saturated matrix solution had to be made freshly every day. For co-crystallization of the sample and matrix 1µl of the matrix solution was carefully mixed with 1 µl of the sample with the pipette. 1 µl of the mixture was applied carefully onto MALDI target (without touching the target with the tip) and dried in the air at the room temperature.
2.5.6.4 Obtaining the mass spectra on MALDI-TOF
MALDI positive ion mass spectra were obtained with a Bruker Daltonik Reflex III, using 337 nm nitrogen laser, with 200 ns extraction delay. Spectra were obtained as averages of 100 laser shots.
2.5.7 Purification of FGE
FGE was purifed from the medium of HT1080 cells stably overexpressing FGE-His6 in two steps. Cells were grown to near confluency in normal medium and after 48 hr the medium was collected by replaced with fresh medium. The medium was spun down at 2,500 rpm (JA10 rotor) for 30 min and the supernatant was precipitated by 50% ammonium sulfate.
Ni-NTA purification
Five liters of the precipitated medium was centrifuged at 10,000 rpm/ 30 min/
4oC. Resuspended the pellet in 35 ml of buffer A. Dialyzed the suspension against the buffer A (2.5 litre) overnight. Next day dialyze again with fresh buffer A for 4 to 6 hr. The dialyzed material was cleared by ultra-centrifugation at 35,000 rpm/
30 min/ 4oC and the supernatant was subjected to Ni-NTA affinity chromatography.
Buffer A: 50 mM NaH2PO4, 300 mM NaCl pH-8.0.
Flow through was collected from the Ni-NTA column and the matrix was subjected to washes and eluted as follows.
Buffer No. of times Volume Incubation time
and temperature
Wash (W1) Buffer A 6 15 ml
Wash (W2- W7) Buffer A with 25 mM Imidazole 6 30 ml 10 min, 4oC
Elution (E1-E3) Buffer A with 100 mM Imidazole 3 15 ml 20 min, 4oC
Elution (E4) Buffer A with 250 mM Imidazole 1 15 ml 20 min, 4oC
46 Chapter2. Materials and Methods
Regeneration of the column 1% SDS 1 15 ml 20 min, 80oC
and washed with water thoroughly
For further purification of FGE-His6 elutions were pooled and subjected for second chromatography purification using C69S peptide as a ligand. Elutions from Ni-NTA purification were incubated with C69S affigel 10 matrix (35 ml) overnight at 4oC.
Buffer No. of times Volume Incubation time and temperature
Wash (W1 W6) Buffer 1 6 50 ml
Wash (W7) Buffer 2 1 50 ml
Elution (E1) Buffer 5 1 35 ml 4 hr, 4oC, on rotator
Elution (E2) Buffer 4 1 35 ml
Elution (E3) Buffer 5 1 35 ml over night, 4oC, on rotator
Elution (E4) Buffer 4 1 35 ml
Elution (E5) Buffer 5 1 35 ml 4 hr, on rotator
Elution (E6) Buffer 4 1 35 ml
Regeneration of the column 1% SDS 1 35 ml 30 min, 80oC
and washed with water thoroughly and store with 0.02% NaN3 in 50% ethanol
Elution fractions are pooled and concentrated by using ultra-thimble (UH 100/25, Schleicher & Schuell). The concentrated sample is dialyzed against 10 mM Tris-HCl, 2.5 mM DTT pH-8. Hence the FGE purified was checked for the purity by Coomassie staining and activity by in vitro FGE assay.
2.5.8 In vitro FGE activity assay
For monitoring the activity of FGE, the N-acetylated and C-amidated 23-mer peptide P23 (MTDFYVPVSLCTPSRAALLTGRS) was used as substrate. The conversion of the cysteine residue in position 11 to FGly was monitored by MALDI-TOF mass spectrometry. A 6 µM stock solution of P23 in 30% acetonitrile and 0.1%
trifluoroacetic acid (TFA) was prepared. Under standard conditions, 6 pmol P23 were incubated at 37oC with 2-5µl enzyme (purified FGE, placenta reticuloplasm, FGE-His6or∆34-68 FGE-His6) in a final volume of 30µl 50 mM Tris/HCl (pH 9.0), containing 67 mM NaCl, 15 M CaCl2, 2 mM DTT. To stop the enzyme reaction, 2µl 10% TFA was added.
Matrix preparation for MALDI-TOF α-cyano-hydroxycinnamic acid: prepared a saturated solution of α-cyano in 100% acetone and vortexed, sonified and centrifuged before use. Nitrocellulose: solubilize nitrocellulose(BioRad 162 0145)
2.5. Biochemical Methods 47 in 50% acetone, 50% isopropanol to a concentration of 10 mg/ml. α-cyano/nc matrix: mix 4 volumeα-cyano with 1 volume of nitrocellulose.
Sample preparation for MALDI-TOF Pipette 0.5 µl α-cyano/nc matrix onto the target (polished steel) let it to dry Add 0.5 µl 10% formic acid and 1 µl of the reaction mix and let it to dry.Wash twice with 2µl 5% formic acid and let it to air dry.
MALDI-TOFmass spectrometry was done with a Reflex III (Bruker Daltonics) us-ing reflectron mode and laser energy just above the desorption/ionization threshold.
All spectra were averages of 200-300 shots from several spots on the target. The mass axis was calibrated using peptides of molecular masses ranging from 1000-3000 Da as external standards. Monoisotopic MH of P23 is 2526.28 and of the FGly containing product 2508.29. Activity (pmol product/hr) was calculated on the basis of the peak height of the product divided by the sum of the peak heights of P23 and the product.
2.5.9 Determination of FGE activity in cells
The trypsinized cells were washed with phosphate-buffered saline (PBS) and resuspended in 50 mM NaH2PO4, 300 mM NaCl and 10mM Imidazole buffer. The cells were lysed by ultrasonication and cleared by centrifugation at 125,000 Xg for 30 min. The supernatant was subjected to Ni-NTA chromatography according to the Qiagen instruction manual. FGE activity in the eluted fraction was measured peptide as described above.
2.5.10 Galactose-6-Sulfatase activity assay
Activity of ASA, STS, and galactose-6-sulfatase were determined as described [Rom-merskirch and von Figura,1992].
2.5.11 Gel filtration (size exclusion) chromatography on superdex 200 column on the SMART system
HT1080 cells stably expressing galactose-6-sulfatase and FGE-His6 or∆34-68 FGE-His6 were grown to near confluence in 100-mm dishes. Cells were harvested by scrapping and lysed in 2 ml of PBS by ultrasonication. The cell lysate was cleared by centrifugation at 125,000 X g for 30 min at 4oC. 50µl of supernatant was subjected
48 Chapter2. Materials and Methods to Superdex 200 PC 3.2/30 column (Pharmacia) on the SMART system. Prior to the run the column is equilibrated with the running buffer (1X PBS). Proteins were eluted with the same buffer at a flow rate of 0.04 ml/min. Fractions of 0.08 ml were collected. Proteins were separated by 15% SDS-polyacrylamide gel and analysed by Western blot using a mixture of galactose-6-sulfatase and His-tag monoclonal antibodies.
2.5.12 Co-immunoprecipitation
For co-immunoprecipitation of Gal-6-S and FGE-His6 or ∆34-68 FGE-His6 the co-eluted fractions were incubated with FGE antibodies for overnight at 4oC (see above section). To the suspension prewashed 10% StaphA (PANSORBIN cells) was added, and the sample was incubated for 1 hr at 4oC. Centrifugation to pellet down the StaphA, antibody-antigen complex followed by washes with 1X PBS for five times.
Elution by boiling the pellet with antibody-antigen complex in 1x Laemmli buffer, and proteins are separated by SDS-PAGE. Analysis by Western blotting using a mixture of galactose-6-sulfatase and His6-tag monoclonal antibodies, followed by peroxidase-conjugated anti-mouse as a secondary antibody. Detection by using a chemiluminescent substrate.
2.5.13 Metabolic labeling and Immunoprecipitation
HT1080 rtTA cells were grown to 50-60% confluency transiently transfected with wt-pFGE , truncated pFGE (∆PGEL) and pFGE with KDEL. After 6 hr of transfection, medium was replaced by normal medium with 0.5 mg/ml Doxycyline. After 12 hr of induction, cells were starved in 2 ml of cysteine-free DMEM for 1 hr, and pulsed for 90 minutes with 1 ml of medium containing 100 mCi of 35S-labeled cysteine.
Cells & medium were harvested after pulse (0 hr), cells by scraping after washing with phosphate buffered saline and culturing in 1 ml of chase medium (DMEM with 10% FCS, 1% pencillin/streptomycin) for 3 hr and 6 hr. Immunoprecipitation of pFGE from cells and medium was performed with pFGE antiserum according to a protocol described earlier [Gieselmann et al., 1992]. Pellets were finally solubilized and subjected to SDS-PAGE and phosphorimaging (BAS1000, Raytest).
Densitometric quantification of pFGE was done using the MacBAS software (Fuji).
HT1080 FGE-His6 or ∆34-68 FGE-His6 stable cells were grown to 80% conflu-ency. cells were starved in 2 ml of cysteine-free DMEM for 1 hr, and pulsed for 90 minutes with 1 ml of medium containing 100 µCi of 35S -labeled cysteine. Cells
2.5. Biochemical Methods 49
& medium were harvested after pulse (0 hr), cells by scraping after washing with phosphate buffered saline and culturing in 1 ml of chase medium (DMEM with 10% FCS, 1% pencillin/streptomycin) for 4 and 8 hr. Immunoprecipitation of pFGE from cells and medium was performed with FGE antiserum according to a protocol described earlier [Gieselmann et al., 1992]. Pellets were finally solubilized and subjected to SDS-PAGE and phosphorimaging (BAS1000, Raytest). Densitometric quantification of FGE was done using the MacBAS software (Fuji).
2.5.14 Preparation of reticuloplasm from human placenta
Human Placenta tissue was obtained from New Bethlehem hospital directly after the delivery to ensure the freshness of the material. Placenta was transported on ice.
Immediately upon arrival the placenta was washed several times in wash buffer (10 mM Tris, pH 7.5; 150 mM sodium chloride). All the following steps were performed at 4oC. One preparation was made from 70 g of placenta. Placenta was cleaned thoroughly to remove blood and connective tissue and thoroughly chopped with razor blades. Chopped placenta was mixed with 3 volumes of buffer A (Protease inhibitor mix 1:500). The mix was homogenized with the help of boring machine in a Teflon potter. The homogenized material was pelleted by centrifugation in Falcon 50 ml tubes by 500 g for 10 min. The supernatant was filtered through the gaze, transferred into a new tube and subjected to the centrifugation by 3,000 g for 10 min. The resulting supernatant was centrifuged again in JA 20 tubes in JA20 rotor for 10 min by 20,000 g. The supernatant was used subjected for the following centrifugation step. The final centrifugation step was performed in Ti 45 or Ti 60 rotors (Beckmann). The polycarbonate tubes for corresponding rotors were half filled with the supernatant and the cushion buffer was carefully under-layered to the bottom of the tube. Prepared tubes were centrifuged in the corresponding rotors for 3.5 hours at 45,000 rpm (Ti 45 rotor) or 3 hours at 50,000 rpm (Ti
Immediately upon arrival the placenta was washed several times in wash buffer (10 mM Tris, pH 7.5; 150 mM sodium chloride). All the following steps were performed at 4oC. One preparation was made from 70 g of placenta. Placenta was cleaned thoroughly to remove blood and connective tissue and thoroughly chopped with razor blades. Chopped placenta was mixed with 3 volumes of buffer A (Protease inhibitor mix 1:500). The mix was homogenized with the help of boring machine in a Teflon potter. The homogenized material was pelleted by centrifugation in Falcon 50 ml tubes by 500 g for 10 min. The supernatant was filtered through the gaze, transferred into a new tube and subjected to the centrifugation by 3,000 g for 10 min. The resulting supernatant was centrifuged again in JA 20 tubes in JA20 rotor for 10 min by 20,000 g. The supernatant was used subjected for the following centrifugation step. The final centrifugation step was performed in Ti 45 or Ti 60 rotors (Beckmann). The polycarbonate tubes for corresponding rotors were half filled with the supernatant and the cushion buffer was carefully under-layered to the bottom of the tube. Prepared tubes were centrifuged in the corresponding rotors for 3.5 hours at 45,000 rpm (Ti 45 rotor) or 3 hours at 50,000 rpm (Ti