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.