SDS PAGE ( from David Morgan 1990)

General: Separation of protein by size, under reducing or nonreducing conditions. The percentage of acrylamide used depends on the size of protein.

Solutions:

1) 30% Acrylamide for SDS gels (store at 4⁰C) 500ml Buffer

- 29.2% Acrylamide -146g

- 0.85Bisacrylamide -4g

- solvent is ddH₂O

Note-Filter through Whatman #1 paper

2) Upper gel buffer, pH 6.8 (store at 4⁰C) 500ml

- 0.5 M Tris base -30.28g

- 0.4% SDS -2 g

Note-pH before adding SDS

- bring to Ph 6.8 with conc. HCl(about 8ml) The pH gradient is very steep near pH 7(don't back titrate)

3) Lower gel buffer, pH 8.8 (store at 4°C) 500 ml

-1.5 M Tris-base -90.82 g

-0.4 % SDS -2 g

Note-pH before adding SDS

• bring to pH 8.8 with conc. HCl(about 8ml) (don't back titrate)

4) SDS sample buffer pH 6.8 (store at 4⁰C) 100ml 1x 4x

-62.5 mM Tris base -6.25ml 1M 12.5 2M

-10%(v/v) glycerol -10ml 40ml

-5%(v/v)b-mercaptoethanol -5ml 20ml

-2.3%SDS -2.3g 9.2g

-10% v/v Bromphenol Blue -10ml 0.01g

Note-pH before adding SDS

5) Running buffer(1x) (do not pH, store at RT) 1L 10x

-25mM Tris Base -30.28g

-192mM glycine -144.13g

-o.1%SDS -10g

6)Bromphenol Blue (store at RT) 50ml

-0.1%bromphenol blue -0.05g

-10mM Tris pH 6.8 -0.5ml 1M stock

7)Coomassie Blue (conc.) 500ml

-0.1%Sigma brilliant blue R -0.5g

-20% Methanol -100ml

-7% Acetic Acid -35ml

8) Destain (store at RT) 4L

-20% Methanol -800ml

-7.5% acetic acid -300ml

-H₂O -2.9l

9) Running Gel Solution 10% 7.5% 12.5% 15%

-ddH₂O -6.7ml -8.0ml -5.3ml -4ml

-lower gel buffer -4ml -same -same -same

-30% Acrylamide -5.3ml -4ml -6.7ml -8ml

then add

-10% NH₄ persulfate -25ml -same -same -same

-TEMED -12.5ml -same -same -same

Note: To make other % gels only change the amount of acrylamide and ddH₂O. For X% gels:

-vol.(mls) of 30% Acrylamide=X(desired gel %)x 16ml/.3

-vol.(mls) of ddH₂O=10mls-vol.of 30%Acrylamide

-For Minigel make 8mls total with the same proportions

10) Stacking Gel Solution (enough for 2 gels)

-2.5ml upper gel buffer

-5.9ml ddH₂O

-1.6ml 30% Acrylamide

then add

-30ml 10% Amonium persulfate

-10ml TEMED

Note: The stacking gel does not change with changes in the percent of the running gel.

Protocol:

1. Clean glass plates, spacers, combs and tubing with 95% ethanol

2. Place spacers on the edge of the notch parallel to the sides of the plates and about 1 cm from the bottom

3. Lay the tubing across the bottom of the glass about a 1/2 centimeter below the spacers and upward along the outside edge of each spacer.

4. Place the other plate on top and straighten yhe tubing with the comb

5. Place this sandwich in the gel box with the notch facing the box and the???????