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Wednesday, April 27, 2011

SDS PAGE : Protocol


SDS-PAGE - Protocol

SDS-PAGE

SDS PAGE - Sodium dodecyl sulphate polyacrylamide gel electrophoresis, is an electrophoretic technique used to separate out proteins based on the molecular weigtht.

1. The electrophoresis apparatus was assembled by taking two glass plates (Bangalore Genei), spacers are kept in between the two plates, bottom is sealed with sealing gel.

Electrophoretic Unit  
 

2. The stock solution :
a) 0.5M Tris (pH 6.8)
- 6.05 g tris in 100ml distilled water; pH was adjusted with conc. HCl
b) 1.5 M Tris (pH 8.8)
- 18.15 g Tris in 100ml distilled water; pH was adjusted with conc. HCl
c) 10% SDS
- 1g in 10ml 
 
3. The working solution :
a) Solution A (30%)
Acrylamide – 29.2 g
Bisacrylamide – 0.8 g
b) 10% APS – 100mg in 1ml
c) electrophoresis buffer (1L)
SDS – 1g
Tris – 3g
Glycine – 14.4g
4. Sampling buffer :
I. reducing -> a) 0.5 Tris (pH 6.8) – 0.5ml
b) glycerol – 1ml
c) ß mercaptoethanol – 1ml
d) SDS – 0.2ml
e) Bromophenol Blue (BPB)– a pinch
II. non-reducing -> a) 0.5M Tris (pH 6.8) – 1ml
b) glycerol – 1.5ml
c) SDS – 0.2g
d) Bromophenol Blue (BPB)– a pinch
5. Separating gel (12%) :
a) distilled water – 2.8ml
b) Tris – 1.25ml
c) acrylamide – 0.83ml
d) SDS – 75 µl
e) APS – 52.5ml
f) TEMED – 6 µl
6. stacking and sealing gel (same composition for both) :
a) distilled water (D/W)– 2.8ml
b) Tris – 1.25ml
c) acrylamide – 0.83ml
d) SDS – 50 µl
e) APS – 50 µl
f) TEMED – 5 µl
7. The lower portion of the assembly sealed by using the sealing gel. The separating gel was poured in between the plates and allowed to solidify. After solidification the stacking gel was poured and placed the comb into the stacking gel for making wells. Allow it to solidify. After solidification the electrophoresis buffer was poured into the apparatus. The sample was added into the wells. 
 
Sample Preparation :
Mix 20ul of sample with 5ul of sample buffer
BSA : 1mg/ml - 10ul + 10ul DW + 5ul of sample buffer
Protein Mol.wt Marker - see the provider catalogue and dilute accordingly.
Vortex all the samples, boil @ 100 ºC for 15 mins. and centrifuge for 2-3mins.
Load the samples into wells and note down the order of loading.
8. Switch on the Electrophoretic power supply with 160v, 40mA electricity, run the gel till it touches the sealing gel. After separation of bands, switch off the current and remove the gel carefully for staining.

 CBB ( Coomassie Brilliant Blue ) staining : CBB R-250
a) CBB – 0.25g
b) methanol – 50ml
c) acetic acid – 10ml
d) D/W – 40ml
Dip the gel in CBB stain and leave it on a gel rock for a few mins.
CBB Destaining :
a) methanol – 50ml
b) acetic acid – 10ml
c) D/W – 40ml
Keep the gel in destaining solution in rocker, destain till the protein bands are clearly visible.


 

A CBB Stained Gel
 
OR 

Silver staining :
- wash the run gel with D/W.
- rock with 50ml of 50% methanol for 15 mins.(twice)
- rinse once with D/W for 10 seconds.
- wash with silver solution for 15 mins.
- wash 5 times with D/W (1 min. duration each)
Silver staining solution :
a) add 200mg of AgNO3 in 50ml of D/W.
b) add 1ml of 1M NaOH and mix the solution.
c) add 650 µl of liquid ammonia and add immediately to the gel. Shake for 15mins. 

Developing solution:
a) add 250 µl of 1% citric acid.
b) 50ml of water
c) 50 µl of formaldehyde. 

 

A Silver Stained Gel


Note: Some follow slightly different procedure in buffer making and sample preparation.