Protein overexpression and induction in E. coli
SDS-PAGE
Determination of the protein b/w in the pellet and soluble state
Protein purification using Ni-chelated resin
SDS-PAGE
Coomassie staining
Examination of protein purification
Western blotting
SDS-PAGE (polyacrylamide gel electrophoresis): denatured gel
Polyacrylamide gel:-SDS: native gel
+SDS:denatured gel
reducing reagent: beta-mercaptoethanol, DTT (dithiothreitol)
SDS-PAGE separates protein samples by the mobility of each macromolecule depending
on the linear length of proteins’ primary structure and its mass-to-charge ratio.
2.475 ml
1.575 ml
62.5 ul
(Ammonium persulfate)
Tetra-methyl-ethylene-di-amine
62.5 ul
5 ul
Protein loading (sample) buffer
4X stock for protein sample buffer
2.0 ml
1M Tris-HCl pH 6.8
0.8 g
SDS
4.0 ml
100% glycerol
0.4 ml
14.7 M β-mercaptoethanol
1.0 ml
0.5 M EDTA
8 mg
bromophenol Blue
• SDS contained in the sample buffer makes proteins negatively
charged proportionally to their length.
• 2-mercaptoethanol/DTT breaks disulphide bonds.
• Staining solution
0.1% Coomassie Brilliant Blue R-250
50% methanol
10% glacial acetic acid
D.W
• Destaining solution
40% methanol
10% glacial acetic acid
D.W
• Staining an SDS-PAGE gel with Coomassie blue
1. Stain a gel in staining solution for 20 min with gentle agitation.
2. Destain a gel in destaining solution. Replenish the solution several
times until background of the gel is fully destained.
3. Check the expressed protein in a destained gel
4. Store the destained gel in D.W
Coomassie blue staining
How to detect protein in an SDS-PAGE?
http://www.youtube.com/watch?v=b1dXzU4iOw
Western Blotting
Flow chart
1. Running a SDS-PAGE gel
2. Protein transfer from SDS-PAGE gel to PVDF
(Polyvinylidene fluoride)/nitrocellulose membrane
3. Western blotting
a. Blocking
b. Incubation with primary antibody
c. Washing
d. Incubation with secondary antibody
e. Washing
f. Chemiluminescence
https://www.youtube.com/watch?v=VgAuZ6dBOfs
Blot onto PVDF membrane (Transfer)
1.
2.
3.
4.
5.
cut PVDF membrane to size = 8 x 6cm
place a blotting cassette black side down in a large box of cold blotting buffer
immerse 2 blotting pads and a piece of Whatman paper (x2)
wet this in methanol & then equilibrate in blotting buffer
assemble the blotting sandwich (smooth out any air bubbles at each stage)
• Black side of cassette
• blotting pad
• a piece of Whatman paper
• resolving gel (stacker has been removed)
• PVDF membrane
• 1 piece of Whatman paper
• blotting pad
• white side of the cassette
6. place in blotting module in running tank
7. if only one gel is to be blotted then fill the other space with a second cassette containing
just two blotting pads
8. insert a cooling block containing frozen water
Transfer buffer (blotting buffer)
9. fill tank with transfer buffer & place in an ice bucket
48 mM Tris base
39 mM glycie
10. run at 100V for 1 hour
20% Methanol
Fill up D.W up to 1 litre
1. Blocking
Place blot in 5% Skim milk in TBS-T for at least 1 hour
rinse blot in TBS-T, then wash in fresh TBS while preparing the primary antibodies
2. Primary antibody prep:
Dilute primary antibody by a factor that fits in a 15 ml conical tube with 5% skim milk in
TBS-T (dilution factor is depends on type of antibody & manufacturer recommendation )
Incubate primary with shaking in a clean container for at least 1 hour or o/n
3. Wash the membrane with TBS-T
5 min in TBS-T x 2
10 min in TBS-T x 2
15 min in TBS-T x 1
4. Secondary antibody (conjugate with Horseradish Peroxidase (HRP) attached, in fridge)
1/2000 (it is changeable) in 5% skim milk in TBS-T
use 10 ml per blot and incubate with shaking for at least 1 hour
5. Wash the membrane with TBS-T, then:
5 min in TBS-T x 2
10 min in TBS-T x 2
15 min in TBS-T x 2
http://www.piercenet.com/method/chemilumi
nescent-western-blotting
Detect the signal by chemiluminescence
1. Place some OHP film in a light resistant cassette
2. Using forceps grasp a edge of the membrane, remove excess liquid, and
place on OHP film
3. mix 0.5 ml of each of HRP reagents 1 and 2 together per blot (once
these are mixed then the light emitting reaction starts)
4. working quickly, pipette one ml onto each blot ensuring the entire
surface is covered
5. leave for 1 min
6. place a second piece of film on top and smooth out any air bubbles
7. In dark room expose blot to X-ray film and develop - exposure times of
1 min and 5 min are good starting point (more exposure time is
available, if the signal is weak)
Development of the X-ray film
After exposure X-ray film soaking in developer solution – water – fixer –
water (you can see the band on film at this point)
Oxidation
Luminol
3-aminophthalate
Horse radish peroxidase (HRP) subustrates:
Luminol
 Light
TMB, DAB, ABTS  Coloured products
Alkaline phosphatase (AP) substrates:NBT/BCIP