vers proteins in a net negative charge on of 2-mercaptoethanol reduces disulphide bonds an g is used to further denature proteins. grate in gel according to mass + + + + + + - - - - - - Before SDS Charged R groups H H - - - - - - - - - - - - - - - Hydrophobic areas
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. -. -. -. -. -. -. -. - PowerPoint PPT Presentation
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SDS covers proteins in a net negative chargeAddition of 2-mercaptoethanol reduces disulphide bonds and Boiling is used to further denature proteins.
Migrate in gel according to mass
+
+
+
+
+
+
--
-
-
-
-
Before SDS
Charged R groups
H
H
-
-
--
--- -
-- -
--
--
Hydrophobic areas
Proteins are separated in a ‘discontinuous’ system.
Stacking gel has looser pores to allow proteins to line up first.
Western blots- Ab used to identify Ag immobilized on nylon
SDS PAGE gel separates proteins present in a sample
All proteins are covered withnegatively 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 sampleProteins separate according to charge, size, shape
Silver stain
IgM serum
Western blot
mAb detects light chain
Ig serum
Bromage, E. Comp Biochem Physiol B Biochem Mol Biol. 2006 Jan;143(1):61-9. Epub 2005 Dec 1.
What does a Western blot tell you that a proteingel does not?
Protein blotting
Two major factors affect the efficiency
1. The elution from the gel-use the lowest percentage of acrylamide that willallow resolution-high molecular weight proteins blot poorly
2. Efficiency of binding to the membrane- nitrocellulose (not covalently bound)- Polyvinylidene fluoride (PVDF)- Activated nylon
Transfer of proteins to the membrane
Western blotting-wet transfer apparatus
Western blot-semi-dry transfer of proteins
DetectionPrimary antibody followed by:Radioactive-labelled125I staphlococcal protein A or streptococcal protein G
Chemiluminescent detection-HRP catalyzes the oxidation of luminol in hydrogen peroxide. Luminol decays by light emission.
AP catalyzes the dephosphyorylation of adamantyl-1-2-dioxetanephosphate, resulting in emission of light.
Can see proteins that are not normally visible
Far western technique
Detection of protein-protein interactions using a labelled bait protein
Figure 7 Distribution of the 52 kDa protein in various mouse Figure 7 Distribution of the 52 kDa protein in various mouse tissues as analysed by Southwestern blot analysistissues as analysed by Southwestern blot analysis