Introduction to Biochemistry

Ig Depletion from Foetal Calf Serum (FCS)

1. Introduction
This protocol describes the use of Amersham-Pharmacia 1ml HP protein G columns for the purification of monoclonal antibodies (mAb). Scale-up to the use of 5ml columns is simple (5x flow rate etc.). There are some important considerations ...

A. In general a single column is used for the purification of only one mAb, to eliminate possible cross-contamination. If this is not possible for reasons of expense, the optional step of cleaning the column with guanidine hydrochloride should be used between purifications of different antibodies.

B. The bovine IgG in culture media containing FCS will be co-purifiued along with the mAb. Therefore it is essential to use either serum-free media, or media containing FCS that has been depleted of protein-A-binding Ig. (See separate protocol for pA/pG depletion of FCS).

2. Related documentation
See Amasham protocol that comes with the columns, and on which this protocol is based.

3. Materials
3.1 Biorad peristaltic ECONO pump and appropriate tubing (1.6 mm diameter pump tube).
3.2 Filter unit (Nalgene, MF75 series, polystyrene housing, 0.2um PES membrane: 250ml capacity Cat no 168-0020, 500ml capacity Cat no 166-0020) (can use 0.45um filter and alternatives).
3.3 Hi Trap Protein G HP column, 1ml (Amersham, Cat no 17-04040-01).
3.4 PBSa sterile (IAH, microbiological services).
3.5 0.22uM syringe filter (Millipore, Cat no SLG033RS).
3.6 0.45uM syringe filter (Millipore, Cat no SLHA025OS).
3.7 500ml sterile conical flasks.
3.8 200-400ml beakers for waste collection, lL cylinder for dialysis.
3.9 10ml syringe to pre-soak syringe filters.
3.10 Pipetteman and filter tips.
3.11 Retort stand and clamps.
3.12 1.5ml eppendorf tubes for collection of elution fluid.
3.13 0.1M Glycine pH 2.7.
3.14 1M Tris-HCl pH 9.
3.15 70% ethanol.
3.16 20% ethanol (made with sterile water).
3.17 pH paper.
3.18 Vacuum pump.
3.19 Stopwatch.
3.20 Sticky tape.
3.21 Float-A-Lyser (Spectrum laboratories, 5ml sample volume, Cat no 235054).
3.22 Acrodisc Syringe Filter 0.2µm Low Protein Binding (PALL Gelman Laboratory, product no 4454).



4. Responsibilities

4.1 Personnel using this method / protocol are responsible for ensuring that they have read and understood the contents, and the procedure is followed. They must be suitably trained and competent, as documented in their training records. It is the duty of the Head of Department / Group (or nominee) to ensure that staff are aware of this responsibility.

4.2 Health and Safety: This method / protocol should be used in accordance with the current Health and Safety Policy, and other relevant instructions as issued by the Institute for Animal Health. Due consideration must be given to National standards and regulations.

5. Procedure
VITAL INSTRUCTIONS

Work throughout at +4oC

Although this procedure is done on the bench try to keep it as sterile as possible by cleaning the bench with 70% Ethanol and using good sterile technique.

Always avoid any air getting into the column.

The bottle or container used to fill the column should never empty, it should be set at an angle and tubing should be taped in place to hold secure.

5.1 Stretch the 1.6mm brown/yellow tube around the pump. The tube should be stretched, and the end sections should be right round the back of the pump. The ends will fit into the appropriate slots.

5.2 Turn the pump on (switch underneath pump). Ensure that the direction of pumping is correct.




5.3 Check the ‘diameter of tubing’ setting on the front of the pump is set to 1.6mm.

5.4 Connect a piece of the clear tubing, so that liquid can be fed into the pump. This tubing must ALWAYS be held in place in the bottle or flask using sticky tape. Place this tubing into a 400ml bottle of sterile PBSa and tape in place.

5.5 Ensure the pump is set to run at 1ml/min. Turn the pump on, and run until there is a good sized drop at the end of the brown tubing.

5.6 Connect a 0.45uM and a 0.22uM syringe filter together. Pre-wet the filters using a syringe filled with PBSa. Hold the syringe vertically with the filters on top to ensure the air will be pushed out upwards.

5.7 Connect the filters to the right hand end of the brown tubing (outlet), pushing the filters firmly in place. Ensure the fluid will flow through the 0.45uM filter first then the 0.22uM.

5.8 Attach the appropriate piece of clear tubing to this filter to allow connection of the pump to the column. Pump to fill tubing with PBSa.

5.9 Take the Protein G column from storage and support in a clamp stand.

5.10 Unscrew the black cap from the top of the column and set aside ensuring it is kept as clean as possible. Use a 200ul pipetteman with filter tips to fill the top of the column with PBSa. Push the tip down as far as possible without damaging the column, and ensure you don’t introduce any bubbles.

5.11 Connect the clear tubing to the top of the column. First turn the pump on to allow a drop of PBSa to form at the tip of the tubing, this will ensure no air is taken into the column. TURN THE COLUMN TO ATTACH IT TO THE TUBING. Make sure the seal is good and not leaking.

5.12 Remove the snap-off end of the column outlet, or remove screw cap if re-using a column. Place a waste beaker under column.

5.13 Turn on the pump until PBSa drips from the bottom of the protein G column.

5.14 Switch off the pump and attach a piece of tubing from the bottom of the Protein G column to a waste beaker. The whole of the tubing and column have a volume of approximately 4ml.

5.15 Check all joints in the pump-column train for leaks and tighten/reassemble as needed. Allow 10 column volumes or so of PBSa to run through the column. Collect some of the flow through.

5.16 (Mock-elution to clean column. Not necessary if the column is new or has been stored for only a few days). Switch off the pump and transfer the inlet tubing to a universal containing 2-3ml 0.1M Glycine pH2.7. Switch on the pump. The first 3-4ml of fluid will be PBSa. Collect some of the flow through when the pH of the flow through drops to pH 3.

5.17 Transfer the inlet tube to a universal of PBSa to wash the tubing then back to the bottle of PBSa. Check pH of flow through. Pump until the pH returns to 7. Now pump at least 10ml PBSa to wash column.

5.18 Calibrate the speed of pumping through the column. Attach a 1ml syringe onto the end of the tubing going to waste. Turn on the pump and run until the liquid reaches the 0ml point on the syringe. Now time how long it takes the pump to reach a certain marker in the syringe. If necessary adjust the speed set on the front of the pump.

5.19 Filter the medium through a 0.22uM syringe filter or Nalgene filtration unit (500ml, or appropriate volume) using a vacuum pump to draw the medium though the filter:

• Switch on the vacuum pump and then connect the pump tubing to the filter unit.
• When all the medium has passed through the filter remove the tubing from the filter unit and THEN switch off the pump.
• You never want to turn off the vacuum pump while it is attached to the filter unit as you could get suck back, where dirty air/liquid from pump is sucked into your sterile filtered medium.

5.20 Transfer the inlet tube to the bottle of filtered medium. Pump through the column at 1ml/min. Watch until medium is passing out the outlet tube then transfer the outlet tube so that the flow through collects into a sterile conical flask. Make sure you do not leave the pump unattended towards the end of the pumping period. Otherwise you risk ruining the column by pumping air through it.

5.21 Transfer the inlet tube back to the bottle of PBSa, and wash column collecting to waste. Collect an aliquot of the flow through.

5.22 Before eluting mix 500µl of 0.1M Glycine pH2.7 and 60-200µl 1M Tris-HCL pH 9. Using pH paper ensure this mix is neutralised at pH7. Prepare 10-12 1.5ml eppendorf collection tubes containing an appropriate volume of 1M Tris-HCL pH 9 to neutralise 500µl of 0.1M Glycine pH2.7.

5.23 Switch off the pump and transfer the inlet tubing to a universal containing 3-4ml 0.1M Glycine pH2.7. Switch on the pump. Pump through the column for 90 sec. Place the first elution collection tube under the column, continue to pump whilst collecting 0.5ml of elute every 30 sec. When the Glycine levels get low replace with PBSa.

5.24 Transfer the inlet tube back to the bottle of PBSa and wash the column collecting to waste.

5.25 Pump at least 20 mls of 20% Ethanol through the column. (You can check when Ethanol is emerging to waste by looking for refractive index changes and floating of the eluting fluid).

5.26 Stop the pump and remove the tubing from the bottom of the column. Switch on the pump and hold the bottom seal of the protein G column under the column to allow it to fill with ethonal before attaching loosely while the pump is still running. This will ensure all air is expelled. Turn of the pump and tighten seal. Loosen the top fitting of the protein column (by turning the column), then start the pump and run while removing the top fitting completely. Stop the pump and replace the top seal of the protein G column. Tighten both top and bottom seals as tight as possible by hand before returning the column to its box and to storage at 4oC. WRITE THE DATE, INITIALS AND USAGE ON THE BOX.

5.27 Analyse protein levels in the collected fractions by reading A280. The protein tends to collect in three aliquots.

5.28 Take the peak fractions and transfer to a Float-A-Lyser Dialysis Cassette (or equivalent) to dialise. If using a Float-A-Lyser remove from packaging by holding it firmly at the collar of the floatation disk. Detach the packaging tube and discard the 0.1% sodium azide preservative. Remove the cap and discard the 0.1% sodium azide from inside the membrane. Dispense PBSa inside the membrane to wash and discard. Using a pipette carefully load sample and snap cap back into place. Float in a 1L cylinder of PBSa on a magnetic stirrer. Work at 4oC. Change the PBSa three times over a 24 hour period. After dialysis open the cap and recover sample using a pastette.

5.29 Analyse protein levels by reading A280. Adjust to 1mg/ml with PBSa. If necessary concentrate. Pass through a 0.2µm low protein binding small volume syringe filter to sterilise. Store main aliquot at 40C and open only under sterile conditions. Take a small aliquot and add 10% sodium azide, diluting 1 in 100 to give 0.1% final concentration. This working aliquot can be opened on the bench. Store at 40C.







6. Results

A2801=0.7mg/ml
7. Maintenance
Rinse all pump and associated tubing with 70% Ethanol, drain, and store in the bags provided to avoid contamination.


8. Troubleshooting
Air bubbles before column. You can pump the system backwards for a VERY short time, before the drop at the outlet of the last column reaches the packed medium. This can be used to flush small bubbles from end of the inlet tube. In other case, you will have to dismantle the tubing arrangements and re-prime them. Care must be taken to avoid gravity driven flows by keeping disconnected tubing at appropriate levels. The in-line filters will provide some protection against pumping air onto the columns should the tubing accidentally come out of the source vessel, or should the source vessel become emptied. High surface tension will provide back-pressure preventing flow when the filters are full of air. However, this may not work for high-protein solutions, such as serum, with reduced surface tension, and may lead to explosive disconnection of the tubing under the back-pressure. Therefore this should NEVER be used as a substitute for appropriate surveillance of the column. DO NOT RUN COLUMN OVERNIGHT.

Protein Seperation by Electrophoresis (Hoefer SE400)

1. Introduction
This protocol describes use of the Hoefer SE 400 Vertical Slab Gel Electrophoresis unit to separate proteins, and detection of proteins with coomassie blue staining.

The gel is 14x16cm, the comb is 1.5mm thick with 15 wells and will take a maximum of approximately 80µl.

1D gel electrophoresis under denaturing conditions (in the presence of 0.1% SDS) separated proteins based on molecular size as they move through a polyacrylamide gel matrix towards the anode. After solubilizing all proteins by boiling in the presence of SDS, the protein solution is applied to a gel lane and the proteins separated electrophetically. 2 Mercaptoethanol or DTT is added during solubilization to reduce proteins to their sub units by reducing disulphide bonds.

Detection of protein bands in a gel by coomassie blue staining depends on non-specific binding of a dye, coomassie brilliant blue R, to proteins. The detection limit is 0.3-1µg per protein band. Proteins separated in a ployacrylamide gel are precipitated using a fixing solution containing methanol and acetic acid. The entire gel is then stained with coomassie blue. After destaining blue protein bands appear against a clear background. The gel can be dried to maintain a permanent record.

2. Related documentation
Hoefer SE 400 Series user manual
Current Protocols in immunology

3. Materials
3.1 Alconox or RBS-35 (pierce) or other suitable detergent to clean plates.
3.2 Kaleidoscope prestained broad range standard (Biorad, Cat no 161-0324) or other suitable standard.
3.3 Hoefer SE 400 electrophoresis tank and assessories.
3.4 A suitable power pack.
3.5 30% Acrylamide stock solution.
3.6 1.5M Tris-CL, pH 8.8
3.7 0.5M Tris-Cl, pH6.8
3.8 10% SDS solution.
3.9 10% APS.
3.10 Temed.
3.11 Glycerol.
3.12 2-mercaptoethanol.
3.13 Electrophoresis Buffer (0.025M Tris, 0.192M glycine, 0.1% SDS, pH 8.3).
3.14 Coomassie stain solution (0.05% Coomassie blue, 50% Methanol, 10% acetic acid).
3.15 De-stain solution (5% Methanol, 7% acetic acid).
3.16 Gel dryer.
3.17 3MM paper.
3.18 1-200µl gel tips (Alpha labs, cat no LW1100).
3.19 Small tray for gel staining and de-staining.


4. Responsibilities

4.1 Personnel using this method / protocol are responsible for ensuring that they have read and understood the contents, and the procedure is followed. They must be suitably trained and competent, as documented in their training records. It is the duty of the Head of Department / Group (or nominee) to ensure that staff are aware of this responsibility.

4.2 Health and Safety: This method / protocol should be used in accordance with the current Health and Safety Policy, and other relevant instructions as issued by the Institute for Animal Health. Due consideration must be given to National standards and regulations.

CARE SHOULD BE TAKEN WHEN HANDLING ACRYLAMIDE AND WEIGHING SDS, WEAR GLOVES AT ALL TIMES AND A MASK WHEN HANDLING SDS.
5. Procedure

5.1 Prepare the gel apparatus according to the user manual.
5.2 Prepare an acrylamide resolving gel solution to the required % as follows, add the initiator and catalyst just prior to pouring the gel:

Resolving gel Stacking gel
7.5% 10% 12.5% 15% 4%
Acrylamide stock 7.5ml 10ml 12.5ml 15ml 1.34ml
1.5M Tris-CL, pH 8.8 7.5ml 7.5ml 7.5ml 7.5ml
0.5M Tris-CL,
pH 6.8 2.5ml
10% SDS 0.3ml 0.3ml 0.3ml 0.3ml 0.1ml
Deionised Water 14.6ml 12.1ml 9.6ml 7.1ml 6ml
10% APS 150µl 150µl 150µl 150µl 50µl
Temed 10µl 10µl 10µl 10µl 5µl
Final volume 30.0ml 30.0ml 30.0ml 30.0ml 10ml

5.3 Pipette the gel solution into one corner of the gel plate sandwich, taking care not to introduce any air bubbles. Fill the solution to 3-4cm from the top of the glass plate to allow for 1cm of stacking gel below the wells. Immediately after pouring overlay the gel with a thin layer of water to isolate the gel from atmospheric oxygen during polymerisation. Apply to one corner and allow to flow across the surface unaided.
5.4 Allow the gel to polymerise for a minimum of 1 hour. After polymerisation pour off the overlay.
5.5 Prepare the stacking gel, insert the comb into the sandwich and pour the stacking gel ensuring no air bubbles form under the comb.
5.6 Allow the gel to polymerise for a minimum of 1 hour. Gently remove the comb and rinse out the wells with electrophoresis buffer to remove unpolymerised acrylamide. Drain by inverting the gel sandwich and then fill each well with electrophoresis buffer.
5.7 Prepare liquid protein samples by addition of an equal volume of 2x treatment buffer:


2x sample treatment buffer
0.5M Tris-CL, pH 6.8 0.125M 2.5ml
10% SDS, 0.35M 0.14M 4.0
Glycerol 20% 2.0ml
2-mercaptoethanol 2% 0.2ml
Bromophenol Blue 0.3mM 2.0mg
Deionised water To 10.0ml

5.8 Heat the sample in boiling water for 90 seconds, then allow to cool to room temperature.
5.9 Underlay the prepared sample into the wells using a gel loading pipette tip. Also load suitable markers such as Kaleidoscope broad range standards.
5.10 Attach the upper buffer chamber to the gel sandwich according to user manual.
5.11 Pour approximately 100ml electrophoresis buffer into the upper chamber and inspect for leaks. Fill both chambers to a final volume of approximately 350ml for each chamber. Install the sandwich into the lower chamber and fit the safety lid.
5.12 Run the gel at constant current, at 10 to 20 mA until the samples have passed through the stacking gel. Then increase the current to 20 to 40 mA and run until the tracking dye reaches the bottom of the gel. The gel will take approximately 5 hours to run.
5.13 Once the tracking dye has reached the bottom of the gel, turn off the power supply and disconnect the leads. Remove the safety lid and pour away the buffer from the upper tank down the sink.
5.14 Release the upper chamber and clamps from the gel sandwich. Use the plate separator tool to separate the plates. Carefully lift off one glass plate, remove the spacers and cut away the stacking gel. Remove a nick from the top right hand corner of the gel to allow for orientation.
5.15 Hold the gel over a staining tray and carefully lift one corner so that the gel drops off the plate into the tray. Add enough stain to cover the gel and stain for approximately 1 hour.
5.16 Discard the stain, pour de-stain to cover gel and discard, repeat as necessary to wash away the worst of the stain. Cover the gel with de-stain and leave until the gel has turned clear leaving the protein bands blue. This usually takes overnight.
5.17 Dry the gel to maintain a permanent record.
6. Results
Band pattern for Kaleidoscope standard:

Protein Colour MW on Bis-tris gel
Myosin blue 195815
B-galactosidase magenta 111790
BSA Green 59779
Carbonic anhydrase violet 29888
Soyabean trypsin inhibitor orange 24759
Lysozyme red 12646
Aprotinin blue 6484

7. Maintenance

Clean the gel apparatus after use with water.
Clean the glass plates with a dilute solution of a laboratory cleanser such as Alconox, then rinse thoroughly with water.

8. Troubleshooting
Refer to the Hoefer SE 400 series user manual for troubleshooting.



Reagents:

Running Buffer:
3.025g Tris, 14.4g Glycine, 1g SDS, to 1L with water.

Stain:
25ml Methanol, 37.5ml Acetic acid, 437.5ml water, 0.5g coomassie brilliant blue.

NOTE: DISSOLVE COOMASSIE BLUE IN METHANOL BEFORE ADDITION OF ACETIC ACID AND WATER.

De-stain:
25ml Methanol, 37.5ml Acetic acid, 437.5mml water.

Optimization of machining conditions with practical constrains

http://rapidshare.com/files/32616840/Optimization_of_machining_conditions_with_practical_constraints.pdf

T and B cell isolations

Reagents
Heparin - 1000 U/ml
Ficoll-Hypaque
PBS
RPMI-1640 supplemented with 10 mM glutamine and 15% FBS
AET (0.14M) Dissolve 1.967 g AET in 35 ml di-H2O. Adjust to pH 8.0 with 1.0N NaOH. Bring volume to 50 ml with di-H2O.Store at 2-8oC. Check pH every 2 weeks.
AET-treated SRBC
Wash SRBC 4 times with PBS
Add 4 volumes AET to 1 volume packed SRBC in a 15 m conical tube (1 ml of AET + 0.25 ml packed SRBC).
Mix well. Incubate in a 37oC water bath for 30 minutes. Shake vigorously.
Wash 3 times with PBS.
Store in PBS at 2-8oC for up to 3 days.
SRBC-Absorbed FBS
Mix 10 volumes of FBS with 1 volume packed SRBC.
Incubate at 37oC fir 30 minutes.
Incubate at 2-8oC for 30 minutes.
Centrifuge at 400 g for 10 minutes.
Collect the FBS. Filter sterilize. Store aliquots at -20oC.
Preparation of PBL's
Draw peripheral blood into syringe containing 10 U/ml heparin.
Dilute the blood 1:1 with PBS.
Layer 30 ml of diluted blood onto 20 ml Ficoll-Hypaque.
Centrifuge at 1550 rpm for 30 minutes, room temperature.
Aspirate and discard the supernatant.
Carefully collect the interface of PBL's and transfer into a clean tube.
Fill the tube with PBS. Centrifuge at 1550 rpm for 10 minutes.
Wash the pellet 2 times with PBS.
Count the cells and resuspend to 107 cells/ml in PBS.
Separation of T-Cells
Mix 1 ml of AET-treated SRBC with 10 ml FBS.
Mix and equal volume of PBL's with a 1% (v/v) mixture of AET-SRBC_FBS in a 50 ml tube.
Incubate in a 37oC water bath for 10 minutes.
Centrifuge at 200 g for 10 minutes. Make sure that the cells have pelleted. If not, re-centrifuge for 5 minutes.
Place the tube upright on ice for 60 min.
Layer super over 15 ml of Ficoll-Hypaque leaving 7.5 ml of fluid above the pellet.
Resuspend the pellet by rotating the tube along the long axis.
Stand upright for 1 minute. Remove the top 5 ml and layer on Ficoll-Hypaque.
Rotate as above and transfer to gradient tube.
Wash the tube with 5 ml of PBS and add to gradient.
Centrifuge at 300 g for 40 minutes, room temperature.
Collect the B cells at the interface. Wash 3 times with PBS.
Suspend the SRBC-T cell pellet. Centrifuge at 300 d for 10 minutes.
Aspirate all of the supe. Break up the cell pellet by gently shaking.
Add 9 ml of di-H2O. with shaking for 4 seconds.
Add 1 ml of 10X PBS with shaking.
Immediately fill the tube with 1X PBS.
Centrifuge at 300 g for 10 minutes, and wash 2 times with PBS.

Data Coming Soon..

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