Lab 6. Preparation of rabbit IgG
Enzyme linked Immunosorbent Assay
Variations in the ELISA technique
Used for testing the amount of antibody to an antigen in serum
Add the solution containing antigen to be measured
The sensitivity of the Sandwich ELISA is dependent on four
factors:
1. The number of molecules of the first antibody that are
bound to the solid phase.
2. The avidity of the first antibody for the antigen.
3. The avidity of the second antibody for the antigen.
4. The specific activity of the enzyme attached to the second
antibody.
http://www.chemicon.com/resource/ANT101/a2C.asp
Antibody titres are worked out empirically.
Try several dilutions of antibody
1:250/ 1:500/ 1:1000/ 1:10,000
Dilutions of
known
Standard
Always do duplicates of each test
Cannot just take the Ab concentration
from a different protocol
Ideally the enzyme substrates should be stable, safe and inexpensive.
Popular enzymes are those which convert a colorless substrate to a colored product,
e.g. p-nitrophenylphosphate (pNPP) which is converted to the yellow p-nitrophenol by
alkaline phosphatase.
ELISPOT Assay
E=enzyme
CS-chromogenic
substrate
Kuby 6-11
CP-coloured pdt
Sensitivity of the ELISPOT assay.
The specified number of IFN--producing T cells
was mixed into one million antigen presenting cells
(APC).
The number of IFN--producing cells was measured by
ELISPOT (upper panel) and intracytoplasmic staining
(middle panel); results of ELISA measurements
are shown in the lower panel.
http://www.elispot-analyzers.de
Spot size is proportional to secretion
•High-throughput.
•Fewer cells are required compared to other cellular assays.
•Lymphocytes survive the testing in ELISPOT assays.
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
+
-
+
+ H
+
Hydrophobic areas
-
-
- -
-
-
Before SDS
- --
Migrate in gel according to mass
-
- - - -
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Wikipedia
Range of separation of proteins depends on the
percentage of polyacrylamide used (typical range 12-16%).
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?
http://mullinslab.ucsf.edu/Protocols%20HTML/SDS
_PAGE_protocol.htm
How the discontinuous gel works.
pH 6.8
Glycine
Proteins
ClpH 8.8
Leading ions
Glycine –ve charge
+
Coomassie Blue stained gel
silver staining
Western blots- Ab used to identify Ag immobilized on nylon
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 conditionsSDS and 2-mercaptoethanol are omitted from the gel and sample
Proteins separate according to charge, size, shape
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. 2006 Jan;143(1):61-9. Epub 2005 Dec 1.
Protein blotting
Two major factors affect the efficiency
1.
2.
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
Transfer of proteins to the membrane
Western blotting-wet transfer apparatus
Western blot-semi-dry transfer of proteins
Detection
Primary antibody followed by:
Radioactive-labelled
125
I 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 detectionHRP 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.
Can see proteins that are not normally visible
Far western technique
Detection of protein-protein interactions using a labelled bait protein
Southwestern blot
Figure 7 Distribution of the 52 kDa protein in various mouse tissues as analysed by Southwestern
blot analysis
Biochemical Journal (1998) 329, 623-629 www.biochemj.org