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- | '''Wear gloves at all times!'''
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- | Requirements:
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- | *30% acrylamide(Biorad) '''NEUROTOXIN!'''
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- | *4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8
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- | *4X stacking gel buffer: 0.5M Tris.HCl, 1.92M glycine, 1% SDS water saturated isobutanol
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- | *TEMED( in yellow cabinet )
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- | *10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge
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- | '''Hoeffer Mighty gel system'''
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- | Casting of the gel:
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- | *Per gel take one small glass plate with 'ears', one big white ceramic plate, two grey spacers with perpendicular plastic bits (0.75 mm), and one white comb (0.75 mm)
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- | *Clean the plates etc with soap, rinse with demiwater and ethanol, and dry
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- | *Assemble the system in the gel casting holder. Mark the line of separation/stacking
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- | *Mix the separation gel in 10 ml plastic tube:
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- | '''for 2 gels'''
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- | '''Seperation gel (0.75 mm)''' ''' 12.5%''' '''16%'''
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- | 30% acrylamide 4 ml 5.1 ml
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- | 4X separation buffer 2.4 ml 2.4 ml
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- | MQ 3.2 ml 2.1 ml
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- | 10% APS 28 μl 28 μl
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- | TEMED 28 μl 28 μl
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- | *Pipet the separation gel mix immediately in between the glass plates until the marked line is reached
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- | *Pipet water-saturated isobutanol on top of the polymerizing separation gel
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- | *Let the separation gel polymerize completely before preparing the stacking gel
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- | *When the gel is polymerized, discard the isobutanol and wash the gel with water
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- | *Mix the stacking gel in a 10 ml tube:
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- | '''for 2 gels'''
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- | mQ 1.92 ml
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- | 4X stacking buffer 0.83 ml
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- | 30% acrylamide 560 μl
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- | 10% APS 14 μl
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- | TEMED 7 μl
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- | *Pipet the stacking gel mix immediately on top of the separation gel and place the comb without air bubbles. Mark the teeth of the comb with a marker
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- | *Let the gel polymerize (5-30 min); check also the remaining gelmix in the tube
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- | *Disassemble the gel casting holder and take out the gel/plates
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- | *Use the gel immediately or seal the gel in plastic seal bags and store at 4 °C
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- | '''Assembly of the Tris-Glycine gel in the electrophoresis unit'''
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- | *Take the electrophoresis unit of the Hoeffer system and place it next to a power unit
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- | *Dilute 10X and pour 1X electrophoresis buffer in the container( roughly 1 cm high)
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- | *Place gel in the buffer without air bubbles under the gel
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- | *Use the red clamps to place the gel tightly in the unit
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- | *Pour 1X electrophoresis buffer in the chamber so that the top of the gel is immersed
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- | *Take the comb out of the gel and rinse the wells using a hooked needle and syringe
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- | '''Runninge of the gel'''
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- | *Prepare the samples in 1X SDS sample buffer (NOT nucleic acid loading buffer)
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- | *Boil the samples for 5 min and spin down
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- | *Pipet the samples and protein marker carefully into the wells
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- | *Place the electrode cap on the unit and press lightly.Put the other side of the electrode cables in the correct holes of the power unit
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- | *Switch on the power unit and run until the blue front is appr. 1 cm from the end of the gel. Alternatively, use the prestained protein marker to identify the exact point of stopping
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- | *Disassemble the electrophoresis unit and take the gel
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- | *Take one plate off and cut one corner away for positioning purpose
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- | *Carefully bring the gel into a clean staining container using some demi water and/or spacers
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- | *Stain the gel with CBB or silver
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