1. Specific protein methods

2. Measurement of protein folding
Circular dichroism spectroscopy
Measures the differences in absorption of polarized light
CD is particularly good for
Determine whether a protein is folded and to characterize its secondary and tertiary structure
Comparing structure of proteins obtained from different sources (expression systems)
Demonstrating comparability in solution conformation and thermal stability after changes in the manufacturing processes or formulation

3. Determination of protein secondary structure by CD Secondary structure is determined by CD in the far UV region ( nm). At these wavelengths the chromophore is the peptide bond and the signal rises when it is located in regular folded environment.
Alpha helix, beta sheet and random coil give rise to a chct shape and magnitude of CD spectrum
The signal reflects an average of the entire structure, but it cannot determine the specific residues in the helix portion
The CD spectrum of a protein in the near UV region ( nm) can be sensitive to certain aspects of tertiary structure. Here, the chromophore are the aromatic amino acids and disulfide bonds.
The signal is usually sensitive to the overall tertiary structure
The near UV CD can be very sensitive to small changes in the tertiary structure due to protein-protein interactions or changes in solvent conditions

4. Checking comparability in conformation

5. Checking comparability in conformation
Checking thermal stability

6. PAGE electrophoresis (Poly acrylamide gel electrophoresis)
Electrophoretic separation of proteins
Separation on a polyacrylamide gel matrix by applying an electric field
Gels are cast between a pair of glass plates then polymerization into polyacrylamide chains is done by cross linking
Polyacrylamide is a cross linked polymer of acrylamide.
Cross linker: methylene bis acrylamide
Polymerization: initiated by ammonium persulfate and TEMED (N,N,N,N tetramethylenediamine)
Gel pore size can be varied according to the conc of polyacrylamide (3.5 – 20%) and the cross linking reagent
Gel pore size and electric field influence the rate of movement

7. Proteins could be separated according to their net electrical charge
Another factor for separation is their molecular weight (mass of the protein)
Charge to mass ratio of proteins determine how they are separated in an electric field
If the pH is elevated above the pI of the protein, then the protein is negatively charged and shall migrate towards the anode (native gel electrophoresis)

8. SDS PAGE
In SDS, the detergent sodium dodecyl sulfate is added, SDS ion form complexes with proteins, resulting in an unfolding of the proteins and the amount of detergent complexed is proportional to the mass of the protein.
Proteins in solution with SDS have a net negative charge
Mass to charge ratio remains constant and separation is based on the mass (sieving effect of the polyacrylamide).
Even with high SDS, higher structures remain, addition 2 mercaptoethanol, increase temperature.
Sample buffer: SDS, 2 ME, dye, glycerol, control pH
Load samples
Detection: Coomassie blue, silver stain

10. Enzyme Linked Immunosorbent Assay (ELISA)
WHO????? This course is getting more and more interesting!

11. ELISA Enzyme Linked Immunosorbent Assay (ELISA)
Term Was Coined By Engvall and Pearlmann in 1971
Different Types
Sandwich
Indirect
Similar To RIA, Except No Radiolabel
Can Be Used To Detect Both Antibody and Antigen, qualitative or quantitave
Very Sensitive, pg/mL
Relies on Monoclonal Abs

13. Sandwich ELISA 2 Antibodies Required Must Recognize Different Epitopes
1st Antibody Is Referred To As Capture Ab
2nd Antibody Detection Ab
Enzymes Commonly Used: HRP (Horse Radish Peroxidase) And AKP (Alkaline Phosphatase)
Substrate is TMB (Chromogen)

14. ELISA Plate
96 well plate
Made of plastic on which protein can be adsorbed (bind) easily
Usually done 4C
Special buffer used that will not denature Ab and maximize binding
Blocking step ensures no empty spaces are left
Blocking reagent is often 10% FBS

15. Standard Curve Serial dilutions of the cytokine being measured
Exact concentration is needed
A plot of concentration (pg/mL or ng/mL) is plotted against OD (optical density)

16. Sensitivity Of Elisa
Typically the lowest cytokine concentration that can be detected above negative control
2-3 S.D Above Mean Background Signal
Depending On Antibody Pair Used Sensitivity Varies
Ex. 10 pg/mL

17. Repeat FLASH animation to solidify what they just did.17

18. Ways The ELISA Kit Can Be Used

19. Example: Pregnancy Test

20. Bioassays Assays that identify the biological function
Could be in vivo (animal models)
(Insulin and diabetic animal models)
In vitro: response of a specific receptors in microorgansism, tissue or cell culture
cytokines (IL, IFN)

21. Enzyme assays
Enzyme assays are performed to serve two different purposes:
to identify a special enzyme, to prove its presence or absence in a distinct specimen, like an organism or a tissue
to determine the amount of the enzyme in the sample
A great advantage of enzymes is that they can be identified by their catalysed reactions, in contrast to the other components of the cell, like functional proteins or nucleic acids, which must be determined by direct detection

22. Essential conditions for enzyme assays
A simple but important condition is that substrate and product must differ in the observed feature
Signal to noise ratio must be analysed. the intensity of the signal displayed by the reaction must exceed the noise at least by a factor of two

23. Methods for observing the enzyme reaction
In the simplest case an enzyme reaction can be observed by the appearance (or disappearance) of a coloured compound.
If an enzyme reaction cannot be observed photometrically, other optical methods may be used. Fluorimery. Turbidimetry

24. Influence of the pH on enzyme assays: optimum pH
Two different effects are responsible for this behaviour: (i) the state of protonation of functional groups of amino acids and cofactors involved in the catalytic reaction and (ii) the native, three-dimensional protein structure of the enzyme
Normally the enzyme is fairly stable at its own pH optimum, and so this is recommended not only for testing, but also for storage

25. Buffers and ions
Buffers serve to adjust and stabilize the desired pH during the enzyme assay.
Solvents
According to the cellular milieu water is the standard solvent for enzyme assays
Dependence of the enzyme activity on the temperature
Dependence of enzyme assays on substrates and cofactors