Stock solutions:

• 1.5M Tris pH8.8
• 0.5M Tris pH6.8
• 20% SDS
• 30% acrylamide:0.8% bisacrylamide
  Use milliQ water & filter
• 10% ammonium peroxysulphate (APS)
  store at r.t. in capped tube
• TEMED

5X Sample buffer

• 125mM Tris (pH6.8)
• 5% SDS
• 10% glycerol
• 10mM EGTA
• 0.01% bromphenol blue + 0.01% phenol red
• make 5% ßME prior to use

10X Running buffer: 1.92M glycine / 0.25M Tris-base

For silver staining or MALDI sequencing, it is important to avoid human skin keratin. All solutions should be filtered before use.

Do not, however, filter SDS solutions, they will foam. Add SDS after filtering. It is also a good idea to wear gloves.

Prepare seperating gel
50ml is sufficient for a small gel or a large gel using thin spacers
10ml is sufficient for two minigels

For 50mls use these amounts of acrylamide stock

For 10mls use these amounts of acrylamide stock

For large gels, take 10ml of solution - add 10µL TEMED and use to seal sides and bottom of gel. -- allow 5 minutes to polymerize.

• add 40µL TEMED to remaining solution - pour gel to desired height.
• overlay with water-saturated butanol - allow 5 minutes to polymerize.

Stacking gel (10ml ):

• 1.5 ml acrylamide
  2.5 ml 0.5M Tris (pH6.8)
  100µL APS
  200µL 20% SDS water to 10ml
• Pour and allow polymerization to continue for at least 20 minutes.
• Gel running: make running buffer 0.2% SDS
• run standard gel with 1.5mm spacers at 40-50 mAmps constant current.

Running the gel: The best results are obtained by loading all of the wells (use 1X sample buffer for empty wells) and running relatively slowly, 25-30 mA per 1.5 mm thick minigels.

Running faster impairs resolution.

Molecular Weight Markers: Depends on the source - The invitrogen markers can be used at 5µL per lane for blots and 0.5 µL per lane for silver stained gels.

Stain solution:

• 1.25g coomassie brilliant blue
• 225ml 100% methanol
• 50ml glacial acetic acid water to 500ml / filter through whatman #1 prior to use!

Destain in 5% acetic acid : 10% 1-butanol / cover with plastic wrap.

Coomassie gel staining: overnight with rocking -- you can speed it up by microwaving the gel/stain 3 minutes.

High sensitivity Silver staining protocol

Immunoblot protocol:

• After electrophoresis, cut away teeth of stacking gel and cut gel for staining and blotting -- minigels are 6 x 8 cm .
• Place gel in 20% methanol: running buffer:0.01% SDS and wet blotting paper and nitrocellulose paper.
• if you are using immobilon P -- you have to wet it with 100% methanol - be careful methanol will dissolve nitrocellulose.
• Set up blot cassette - blot at 100V constant volts for 1h or more (1 h should be more than enough however.
• Block blot: 5% low fat dried milk in TBS-Tween(LFDM-TBST) for 15-60 minutes.
  TBS-Tween: 200mM NaCl / 50mM Tris-HCl pH7.4 0.5% Tween
• incubate with first antibody, o/n at r.t. or 4°C in 20% calf serum or in LFDM-TBST.

Ponceau staining of nitrocellulose protocol

Take care to titre antibodies: primary antibodies should be used 1:1000 to 1:10,000 and secondary antibodies between 1:10,000 to 1:40,000.

• wash 3 x 10 minutes each TBS-Tween
• incubate 1-2h in secondary antibody
• wash as above.
• After final washing, mix up ECL reagent (1:1) and incubate with blots for 5 min. typically it takes 2 to 3 ml to cover 1 to 2 minigel blot equivalents.
• pour off the reagent -- seal the bag and expose (protein side up) to X-ray film (XAR5 is fine) -- first test 30 seconds and 5 minutes - develop film
• You can do other exposures if this does not capture the data you need (reaction is stable for about 24 hours).

High sensitivity silver staining protocol

• FIX: 40% ethanol : 12% acetic acid : 50µL 37% formaldehyde / 100ml - overnight
• WASH: 3 times in 50% ethanol
• PRETREAT: 0.02% sodium thiosulfate 100 ml - 1 minute only
• RINSE: 3 times in water (20 seconds each)
• IMPREGNATE: 0.2% silver nitrate + formaldehyde (50 ml water, 0.1 g AgNO3, 37.5 µL37% formaldehyde0 - 20 minutes
• RINSE: 2 times in water (20 seconds each)
• DEVELOP: 100 ml 6% sodium carbonate, 50 µL 37% formaldehyde 4 µL 10% sodium thiosulfate
• STOP: 12% acetic acid
• PHOTOGRAPH: using the coolpix! or you phone (its the new age)