Stacking (Upper) buffer (for the stacking gel)
requires fair bit of HCl so start with ~70mL water when add Tris
0.5M Tris 6.06g pH to 6.8 with 6M HCl until nearing desired pH
0.4% SDS 0.4g
up to 100mL with water
30% Acrylamide*
Acrylamide 30g
Bis 0.8g
up to 100mL with water
wrap aluminium foil around bottle because it is light sensitive
Making SDS Polyacrylamide Gels*
(once the last ingredient, ammonium persulfate, is added, the gel will begin to polymerise)
Separating (lower) Gel - 10% acrylamide (change the proportions of water and acrylamide if different from 10% acrylamide)
Lower buffer 1.9mL
Water 3.1mL
Acrylamide 2.5mL
TEMED 10uL (add TEMED in the fume hood)
Ammonium Persulfate 20uL
Total 7.5mL
*Note that acrylamide in its unpolymerized form is a potent neurotoxin and that gloves must be worn for making gels, setting up the tank for running the gel, and during transferring of the gel
Stacking Gel (for all percentages of lower gel, use this upper gel)
Upper Buffer 1.25 mL
Water 3.25 mL
Acrylamide 0.5 mL
TEMED 10uL (add TEMED in the fume hood)
Ammonium Persulfate 20uL
Total 5 mL
10x Electrophoresis Running Buffer (for the tank)
Tris 121.4g
Glycine 567g
SDS 40g
Water up to 4L
pH 8.3 without adjustment
4x SDS Loading buffer
Glycerol 4mL (put an empty flask onto balance and weight out glycerol by pipetting)
SDS 0.4g
Upper buffer 5mL
up to 10mL with water
� heat while stirring to dissolve the SDS (don�t make the solution boil when heating), and add enough bromophenol blue to make the 1x solution dark enough for easy monitoring when running on a gel
� store at room temperature
10x Towbins Transfer Buffer (for semi-dry transfer of protein from gel to blot)
250mM Tris 15.1g
1.92M Glycine 72.0g
Water up to 500mL
pH 8.3 without adjustment
Amido Black Stain (for viewing all proteins non-specifically after transfer or after blotting)
Methanol 40mL
Acetic Acid 10mL
Amido Black 10B 0.1g
Up to 100mL
Making the gel
1. Use alcohol and Kimwipes to wipe the glass, and then set up the rest of the apparatus
2. For small proteins, use a higher percentage gel
2. Add all ingredients except ammonium persulfate; before adding ammonium persulfate, if the weather is cold in the room, you may wash the solution with hot/warm tap water to help the polymerization
3. Invert tube gently after adding all components
4. Add it up to the correct mark
5. Add t-butanol
6. Remove t-butanol, wash with water, and wipe with filter paper; put in comb
7. Make upper gel and gently shake 2x
8. Add upper gel
9. Remember to keep tubes to check if they�ve polymerised (cap the tube after pouring and see if the material in the tube has polymerised)
10. Label the lanes of the upper gel with a marker if required
Preparing and Loading Samples
1. Set up the gel in the tank and make sure the buffer isn�t leaking (if it is, it means that the glass plates are not slightly protruding up from the screws on both ends)
2. Pour in some running buffer to prevent drying of the gel
3. For each sample, use 1/3 the volume of 4x loading dye (e.g. if have 30uL of sample, add 10uL of 4x loading dye)
4. If running samples in reduced form, add 5% beta-mercaptoethanol in the fumehood
5. once the loading dye has been added, do no place on ice, or the SDS will precipitate out
Running and transferring the gel
1. Check no leaking and it�s running
2. Set for anywhere from 20-30mA, and all the voltage it needs
3. Keep an eye on it to ensure there is current
4. Cut out filter paper and nylon�eight sheets of filter paper for each blot
5. Mark the front of the nylon (make a cut)
FOR NITROCELLULOSE BLOTS DON�T WET WITH METHANOL, OR ELSE IT�LL DISTINTEGRATE IN YOUR HANDS
6. Wet the nylon with methanol
7. Immerse gel, filter paper, and nitrocellulose in transfer solution
(20% methanol + Towbins 1x); use a lower percentage of methanol for larger proteins (e.g. 7%)
8. Proteins in SDS are negatively-charged, so place sandwich as follows
(flatten with each layer except for gel layer because gel is fragile)
3 pieces filter paper
nylon
gel (proteins in SDS are negative)
3 pieces filter paper
9. For transfer conditions, check with manufacturer. We normally set at 20V, 0.300A, 20 minutes (time varies�larger proteins require longer time)
10. Rinse blot with distilled water and hang to dry
11. wipe the transfer apparatus with plenty of water
12 to dry the transfer apparatus more quickly, wipe with some towels, and then fan the remaining liquid with a piece of paper towel
Staining Blot with Amido Black to Detect for Total Proteins Transferred
Note: If desired, perform a Western blot first and then stain with amido black after rinsing away the ECL with water. In other words, if the same blot will be used for both amido black staining and Western blotting, Western blotting should be performed first.
1. Wet a dried blot with methanol if the blot is made with PVDF, or soak in PBS if blot is made from nitrocellulose (note that methanol is toxic, so wear gloves and don�t inhale it)
2. Rinse ~4x with PBS to get rid of the methanol
3. Rinse blot with distilled water once more
2. Invert amido black bottle a few times, and pour some into a petri dish with the blot in it
3. Rock blot a few times
4. Pour amido black back into bottle
5. Rinse blot with distilled water several times to destain the blot
6. Hang blot to dry and place in notebook
If blot turns dark blue irreversibly, dry or put in PBST and then 100% methanol