1. SDS covers proteins in a net negative charge
Addition of 2-mercaptoethanol reduces disulphide bonds and
Boiling is used to further denature proteins.
Charged R groups + - - - + + H - + +
Hydrophobic areas H + - - - -
Before SDS - - - - - - - - - - - - -
Migrate in gel according to mass

2. Proteins are separated in a ‘discontinuous’ system.
Stacking gel has looser pores to allow proteins to line up first.
How does an SDS-PAGE gel really work?

3. Western blots- Ab used to identify Ag immobilized on nylon

4. SDS PAGE gel separates proteins present in a sample
All proteins are covered with
negatively charged SDS and
migrate according to mass
Native PAGE gels run under non-denaturing conditions-
SDS and 2-mercaptoethanol are omitted from the gel and sample
Proteins separate according to charge, size, shape

5. What does a Western blot tell you that a protein gel does not?
IgM serum Ig
serum
What does a Western blot
tell you that a protein
gel does not?
mAb detects light chain
Silver stain
Western blot
Bromage, E. Comp Biochem Physiol B Biochem Mol Biol Jan;143(1):61-9. Epub 2005 Dec 1.

6. Protein blotting
Two major factors affect the efficiency
The elution from the gel
-use the lowest percentage of acrylamide that will
allow resolution
-high molecular weight proteins blot poorly
Efficiency of binding to the membrane
nitrocellulose (not covalently bound)
Polyvinylidene fluoride (PVDF)
Activated nylon

7. Transfer of proteins to the membrane

8. Western blotting-wet transfer apparatus

9. Western blot-semi-dry transfer of proteins

10. Primary antibody followed by: Radioactive-labelled
Detection
Primary antibody followed by:
Radioactive-labelled
125I staphlococcal protein A or streptococcal protein G
Enzyme-linked secondary antibodies
-horseradish peroxidase (HRP)
-alkaline phosphatase-BCIP/NBT
BCIP (5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt) and
NBT (Nitro-Blue Tetrazolium Chloride).
Chemiluminescent detection-
HRP catalyzes the oxidation of luminol in hydrogen peroxide.
Luminol decays by light emission.
AP catalyzes the dephosphyorylation of adamantyl-1-2-dioxetane
phosphate, resulting in emission of light.

11. Can see proteins that are not normally visible

12. Far western technique
Detection of protein-protein interactions using a labelled bait protein

13. Biochemical Journal (1998) 329, 623-629 -
Southwestern blot
Figure 7 Distribution of the 52 kDa protein in various mouse tissues as analysed by Southwestern blot analysis
Biochemical Journal (1998) 329,