laboratory procedure for protien detection and purification

1. C H I N C H U E L E Z E B T H
M T E C H B I O P R O C E S S E N G I N E E R I I N G
M E T ’ S S C H O O L O F E N G I N E E R I N G
M A L A
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2. ELECTROPHORESIS
 Separate and characterize proteins by applying electric current
 Rapid and sensitive
 Polyacrylamide gel is the medium preferred
 Large number of sample analysis possible
 Poly acrylamide gel:
Polymer of acrylamide and bis-acrylamide
Per sulphate ion as catalyst and TEMED as chain initiator
Oxygen act as inhibitor
 Porosity based on relative proportion of monomers
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3. PRINCIPLE
 SDS is an anionic detergent which binds strongly to, and
denature proteins.
Linearize the protein (primary structure)
 SDS can disrupt hydrophobic areas and coat proteins with
many negative charges
 Protein-SDS complex carries net negative charges, hence
move towards the anode and the separation is based on the
size of the protein
 Importance in purification
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6.  Stock acrylamide solution
Acrylamide 30% : 30g
Bis acrylamide : 0.8g
Water : 100ml
 Separating gel buffer
1.875M Tris HCl : 22.7 g
(pH 8.8)
Water : 100ml
 Stacking gel buffer
0.6M Tris HCl : 7.26g (pH 6.8)
Water : 100ml
 Polymerising agents
 Ammonium persulfate 5% : 0.5g/10ml
 TEMED
 Electrode buffer
 0.05M Tris : 12g
0.192M glycine : 28.8g pH 8.2-8.4
0.1% SDS : 2g
Water : 2L
MATERIALS REQUIRED
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7.  Sample buffer
 Tris HCl buffer ph-6.8 : 5ml
 SDS : 0.5g
 Sucrose : 5g
 Mercaptoethanol : 0.25ml
 Bromophenol blue : 1ml
 Water : 10ml
Store frozen in small aliquots
 10% SDS solution
 Protein stain solution
 Coomassie brilliant blue : 0.1g
 Methanol : 40ml
 Acetic acid : 10ml
 Water : 50ml
 Destainer
As above without the dye
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9. PROCEDURE
 Thoroughly clean and dry the glass plates and spacers, then
assemble them properly. Hold and clamp in an upright position
 White petroleum jelly or 2% agar is applied around the edges of
the spacer to hold them in place and seal the chamber between
the glass plates
 Prepare sufficient volume of separating gel mixture
For 15% For 10%
Stock acrylamide
solution
20ml 13.3ml
Tis-HCl (pH-8.8) 8ml 8ml
Water 11.4ml 18.1ml
Ammonium per sulphate 0.2ml 0.2ml
10% SDS 0.4ml 0.4ml
TEMED 20µl 20µl
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10.  Mix gently and carefully, pour the solution in the chamber
between the glass plates
 Layer distilled water on top of the gel and leave to set for 30min
 Prepare stacking gel
 Remove water from top of the gel and wash with stacking gel
solution. Pour stacking gel mixture, place the comb and allow to
set
Stock acrylamide solution 1.35ml
Tris-HCl (pH 6.8) 1ml
water 7.5ml
Ammonium per sulphate
(5%)
50µl
10% SDS 0.1ml
TEMED 10µl
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11.  After polymerisation, comb is removed carefully and install the
gel in the electrophoresis apparatus
 Fill it with electrode buffer
 Connect the cathode at the top
 Sample preparation:
Prepare samples by suitable extraction procedure
Sample heated in boiling water bath for 2-3min with equal volume of 5X
sample buffer and cool to room temperature for complete denaturation and
concentration of protein
 Take required volume of sample and inject into sample well
using micro pipette
 Also load marker sample in a few wells
 Turn on the current to 10-15mA initially and continue to run at
30mA through separating gel until sample reach the
bottom(3/4) of the gel
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13.  After the run is complete, carefully remove the gel from
between the plates and immerse in staining solution for at least
3h or overnight with uniform shaking
 Proteins absorb coomasive brilliant blue
 Transfer the gel to suitable container with at least 200-300ml
destaining solution and shake gently and continuously until the
gel background is colourless (Unbound dye is removed)
 The proteins fractionated into band are seen coloured blue
 The gel can be now photographed or stored in polythene bags
or dried in vacuo for permanent record
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15. PROTEIN MW IN DALTONS
α- Lactalbumin 14200
Trypsin inhibitor soya bean 20100
trypsinogen 24000
Carbonic anhydrase 29000
Albumin, egg 45000
Albumin, bovine 66000
STANDARD MARKER PROTEINS
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