SDS Gel Electrophoresis and Western Blot Protocol

when working with protein, try to use water nanopure

10% SDS
10g SDS
up to 100mL water

Buffer for Gel Preparation
Lower Buffer (to separate the gel)
36.4g of 1.5 M Tris pH 8.8 with 6M HCl until approaching the desired pH
0.4% SDS 8ml of 10% SDS
up to 200ml with water

SDS Gel Electrophoresis and Western Blot Protocol

Compose (Top) buffer (for stacking gel)
requires a fair bit of HCl so start with ~ 70ml water while adding Tris
6.06g of 0.5 M Tris pH 6.8 with 6M HCl until approaching the desired pH
0.4% SDS 0.4g
up to 100mL with water

30% Acrylamide *
acrylamide 30g
bis 0.8g
up to 100mL with water
aluminum foil wrapped around the bottle as a light-sensitive

Make SDS polyacrylamide gel *
(Once the final ingredient, ammonium persulfate, is added, the gel will begin to polymerise)
Splitting (decrease) Gel – 10% acrylamide (acrylamide changing the proportions of water and if it is different from the 10% acrylamide)
Lower buffer 1.9mL
water 3.1mL
acrylamide 2.5ml
TEMED 10ul (add TEMED in a fume hood)
Ammonium Persulfate 20uL
number of 7.5ml

  • Note that in the form of unpolymerized acrylamide which is a potent neurotoxin and gloves should be worn to make a gel, preparing the tank to run gels, and for transferring the gel

Stacking Gel (for all lower percentage gel, using a gel on it)


Top Buffer 1.25 mL
Water 3.25 mL
Acrylamide 0.5 mL
TEMED 10ul (add TEMED in a fume hood)
Ammonium Persulfate 20uL
Total 5 mL

10x Electrophoresis Running Buffer (to tank)
Tris 121.4g
Glycine 567g
SDS 40g
Water up to 4L

pH 8.3 without adjustment

Loading 4x SDS buffer
Glycerol 4ml (put the empty flask to balance and weigh out the glycerol by pipetting)
SDS 0.4g
Buffer over 5 mL
up to 10 mL with water
heat while stirring to dissolve the SDS (not made of boiling solution while heating), and add enough Bromophenol blue to create solutions 1x dark enough for easy monitoring when running on a gel
Store at room temperature

1x Coomasie Blue Dye (for staining gel that is not used to transfer, or to ensure that no protein remaining after the transfer)
Isopropylalcohol 25% 500ml
Acetic Acid 10% 200ml
R250 0.025% Coomasie Blue 0.5g
1.3L water
(Destain is 10% Acetic Acid)
stains 4 hours to ensure all the protein was stained
destain long enough so that the background is clear (a piece of foam or paper towels can be added to the solution to accelerate the destaining)

Towbins 10x Buffer transfer (for semi-dry transfer of proteins from gel to blot)


Tris 250mm 15.1g
1.92M Glycine 72.0g
Water up to 500ml
pH 8.3 without adjustment

10x Phosphate Buffered SalinePBS (to remove methanol from spotting)
1.37 M NaCl to 10x
2g KCl 27mm to 10x
Na2HPO4 80mm to 10x
KH2PO4 20mm to 10x
water to 1L
of 1x PBS pH should be 7.4

PBS TweenPBST (for washing blots)
2L PBS
Tween-20 (Brown bottle) + 1mL by Pipetting in the pipettor or plastic balls
stir half an hour

Amido Black Stain (to see all non-specific protein after blotting)
40ml methanol
Acetic Acid 10 mL
Amido Black 10B 0.1g
until 100mL

Western blot protocol

make gel

  1. Use of alcohol and Kimwipes to wipe the glass, and then set up the rest of the apparatus
  2. For a small protein, using a higher percentage of gel
  3. Add all ingredients except ammonium persulfate; before adding ammonium persulfate, if the weather is cold in the room, you can wash the solution with / warm hot tap water to assist the polymerization
  4. Invert tube gently after adding all the components
  5. Add up the correct sign
  6. Add t-butanol
  7. Remove the t-butanol, washed with water, and wipe with a paper filter; put in comb
  8. Make a gel on and shake gently 2x
  9. Add the gel on
  10. Remember to keep the tube to check whether theyve polymerized (cap after pouring tube and see whether the material in the tube has been polymerized)
  11. Label lanes of gel on the marker if necessary

Preparing and Loading Samples

  1. Set the gel inside the tank and make sure isnt buffer leak (if, 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 gel
  3. For each sample, use 1/3 to 4x the volume of loading dye (for example, if it has 30uL sample, add 10ul of 4x loading dye)
  4. If running the samples in the form of reduced, add 5% beta-mercaptoethanol in the chimney
  5. after loading dye was added, do no place on the ice, or SDS will precipitate

Walking and transferring the gel

  1. Check there are no leaking and running
  2. Set to any of 20-30mA, and all the voltages needed
  3. Keep an eye on it to make sure there is at this time
  4. Cut a sheet of filter paper and filter paper nyloneight for every blot
  5. Mark the front nylon (making the cut a)
    Nitrocellulose blots DO FOR WET WITH METHANOL, OR ELSE IN YOUR HANDS itll DISTINTEGRATE
  6. Moisten nylon with methanol
  7. Immerse gel, filter paper, and nitrocellulose in solution Transfer
    (20% methanol + Towbins 1x); using a lower percentage of methanol for larger proteins (eg 7%)
  8. The protein in negatively charged SDS, so the sandwich place as follows
    (Evens with each layer except for the gel layer brittle gel)
    3 pieces of filter paper
    nylon
    gel (protein in the negative SDS)
    3 pieces of filter paper
  9. To transfer conditions, check with the manufacturer. We usually set at 20V, 0.300A, 20 minutes (time varieslarger protein takes longer)
  10. Rinse the stain with distilled water and hang to dry
  11. The apparatus wash with plenty of water diversion
    12 dry faster transfer apparatus, wipe with a few towels, and then fan the remaining liquid with a piece of paper towel

blotting

  1. Wet the stain dry with methanol if the blot made with PVDF, or soak in PBS if made of nitrocellulose blot (note that methanol is toxic, so wear gloves and Dont inhale it)
  2. Rinse ~ 4x with PBS to remove methanol
  3. Place face blot for blotting
  4. 1% skim milk (made in PBS) 10 min. (Some proteins require a higher percentage of say 5% skim milk and incubation in a cold room for longer eg 30 mins.)
  5. 10 mL total – the primary antibody diluted to appropriate concentration in PBS (+ 0.2% milk) 1 hour
  6. 3x 10 minutes. each PBST
  7. 1: 5000 dilution of the secondary (right pick secondaryeither goat anti-rabbit or goat-anti mouse), 30 minutes
  8. Wash ~ 5x PBST 5-10 minutes each

look

  1. Mix equal amounts of solution A and B of ECL (~ each A & B 1.5ml)
  2. stone manually for 1 minute.
  3. around spotting wrap Saran Wrap
  4. take off the gloves when handling Film
  5. Turn off all the lights (can leave a dim red light