SDS-PAGE Solutions
10x SDS Electrophoresis Buffer, (1 L)
Reference: BioRad Mini Protean II Manual
- 30 g Tris base (fw. 121.1)
- 144 g Glycine (amino-acetic acid, fw 75.07 - IMPORTANT)
- 10 g SDS (or 100ml 10% SDS)
- To 1000 ml with ddH2O (auoclaved purified water)
Check that pH is about 8.6 but DO NOT ADJUST! You've made it incorrectly
if the pH is not close to 8.6
4x SDS Sample Buffer, (50 ml)
(Modified from Hoefer 2x treatment buffer, p. 19 Protein Electrophoresis)
- 8.3 ml 1.5 M Tris, pH 6.8
- 20 ml 20% SDS (if making this, warm to 40C if needed to get into solution)
- 20 ml glycerol
- 3.1 g dithiothreitol (DTT)
- 2.5 ml 1% (w/v) bromophenol blue (BPB) for color
- ddH2O to 50 ml
Label as 4x SDS-sample buffer and store at -2OC (up to 4 months) or -70C (up to at least a year).
1 M Tris pH 6.8, (200 ml)
- 24.2 g Tris base (fw 121)
- about 160 ml ddH2O
- pH to 6.8 with concentrated HCl
- bring final volume to 200 ml
1.5 M Tris pH 8.8, (200 ml)
- 36.3 g Tris base (fw 121)
- about 160 ml ddH2O
- pH to 8.8 with concentrated HCl
- bring final volume to 200 ml
10% SDS
CAUTION: SDS is a strong denaturant and a major-league irritant.
Weigh with caution, when no one else will walk by creating a draft.
- 20 g SDS
- bring final volume to 200 ml with Oasis water
Rapid Coomassie Stain, (500 ml)
(Final concentration is 0.006% Coomassie G250; 10% acetic acid)
Reference: Hoefer Protein Electrophoresis Manual, 1994, p. 40.
- 30 mg Coomassie Brilliant Blue (G-250)
- 450 ml ddH2O
- 50 ml glacial acetic acid
- (Always add the acid to the water!)
Isopropanol Fixing Solution for Rapid Coomassie Staining, (500 ml)
(Final concentration of 25% Isopropanol and 10% acetic acid)
Reference: Hoefer Protein Electrophoresis Manual, 1994, p. 40.
- 325 ml ddH2O
- 125 ml Isopropanol
- 50 ml glacial acetic acid
Old Slow Coomassie Stain, (500 ml)
40% methanol; 10% Acetic acid; 0.025 g Coomassie Blue R250
- 0.025 g Coomassie Blue R250
- 250 ml ddH2O
- 200 ml methanol
- 50 ml glacial acetic acid
- Shake to dissolve, then filter through filter paper
Destain for Old Slow, (500 ml)
- 250 ml ddH2O (50%)
- 200 ml methanol (40%)
- 50 ml glacial acetic acid (10%)
2D Electrophoresis Solutions
The following is a modified procedure that is close to the one that BioRad recommends except Chaps is substituted for the Triton and there is no reducing agent present.
Ratios of ampholytes are as per Anna Gerenday. Detergent is Chaps/NP40 and no reducing agent is present.
4x SDS Sample Buffer (Non-Reducing)
Same as regular 4x SDS Sample Buffer but do not add dithiothreitol (DTT). Can be stored at 4C.
2x IEFSB: First Dimension Sample Buffer
(Using Chaps/NP40 and no reducing agent)
2.76 g urea
330µl Detergent Solution
25µl ampholyte 5-7
100µl ampholyte 3-10
Bring to 5 ml with ddH2O. Warm to ~45C to get into solution.
Make 500µl aliquiots and label with date.
Detergent Solution
(30% Chaps with NP40)
0.3 g CHAPS, 900µl ddH2O. Dissolve, then add 100µl NP40. Store capped tightly at 4C.
Note: If we didn't solubilize in SDS sample buffer, we probably don't need the NP40.
IEF Gel Solution
(As per Protein Iixi 2D instruction manual, p. 14 & like Anna Gerenday's)
2.76 g urea (should be 9.2M final; fw = 60.06)
560µl 40% acrylamide (4.5% final)
50µl ampholyte 5-7
200µl ampholyte 3-10
250µl Detergent Solution
ddH2O to 5ml
Warm gently to <45C with gentle swirling to get into solution
Degas
Then add:
10µl APS and swirl
5µl TEMED and swirl - then pour immediately.
Upper Buffer
(0.1 M NaOH; fw = 40)
2.0 g NaOH
ddH2O to 500 ml
Degas
Lower Buffer
(Final 0.06% Phosphoric acid)
340µl 85% phosphoric acid
ddH2O to 500 ml
Degas
|