NO enzyme, chem rxn. 

Product: Photon of light, chem rxn
2. related of light output to concentration of analyte
3. Acridinium: ester hydrolysis  

Enzyme substrate on Chemiluminescne

Enzyme catalyzed. Product: Light

Greater specifictiy>Chemiluminescene

Has really low detection 

Reaction Dependent

Derivitation 

Light output proportional to analyte Conc

Luciferase assay for ATP 

BInding of Luminescent substance to analyte

Required to separate free from bound

Enzyme immuno assay with perodidase labeled anibody 

Zero order: No change in range

First order: Very little substrate

Increase substrate -> More enzyme active site -> Go to constant -> zero order -> Cant run any faster 

Index of Enzyme Substrate Interaction

Km=1/2 Vmax

What does Km stand for?

High Km: Low affinity for enzyme

Low Km: High affinity for enzyme

1. Kinetic Assay (Repeat max of rate)

2. Continuous Monitoring Method

3. Fixed Period Method 

1. Lag phase

2. Zero order phase.

3. Bend over (Substrate depletion and Product inhibition)

Increase Osmolarity -> Decrease Boiling point

Increase Osmolarity -> Decrease Vapor Pressure

1. Vapor Pressure

2. Freezing Point Depression Osometry 

1. Thermocouple

2. Sample Pad (have specimen on)

3. Button Chamber seal the specimen (express vapor pressure)

1. Chamber is cooled, condensation forms on thermocouple -> stop the cooling process.

2. Chamber temp = Dew Point temp.

Vapor Pressure related to dew point. 

1. If pure water: Osmolarity  zero -> High Vapor pressure. 

2. High Humidity: Lots water inside., high vapor pressure. 

3. Salty Solution: Low vapor pressure, low humidity.  

1. Electrical leads from thermistor (Determine the freezing point)

2. Probe body (Associate with phase changes (Heat release from liquid-> solid))

3. Thermistor 

4. Stirring wire.

Freezing Point Depression Osmometry Characteristics

1. Sample in cell supercooled.

2. Rapid freeze seeded by agitation. 

3. Heat of fusion warms solution to the freezing point. 

How to quantitative the Osmolarity?

1. Deal with substances in the solution. Charges migrate to opposite charge
2.  Ampholute or zwitterion.

3. Isoelectric point (Proteins get many + and -) -> Shift pH to isoelectric so net charge = Zero. 

1. Chambers

2. DC power 

3. Support Media.

4. Buffers 

How does Buffers effect electrophoresis?

What determines the rate of Electrophoresis migration?

1. Charge of molecules

2. Electric Field Strength

3. Friction (Viscosity, MW, Physcial Barriers)

1.Small pores, little one squeeze easily -> Seiving!

2. Big ones have to bounce -> adding more bounds

1. Associated with current flow.

2. Increase heat, increase conductance, increase the rate of migration.

3. Might denature proteins. 

1. Increases the ionic strength of bffer

2. Wick effect 

Increase heat, increase vapor pressure, leave the salt left behind, not vapor. Higher salt concentration when evaporate all water. 
2. This causes osmolarity increases (Osmotic pressure pulls water in both direction) ==>Wick effect => BAD! 

all the salt had been pushed in the middle 

GOAL: Want to separate, spreading the SALT,  

1. Slow down evaporation.

2. Use constant current power source.

3. Refridgerate.

How does Heat effect the current?

V = IR

( Resistance goes down, Voltage drops bc current is constant. The pullls of analyte decrease the separation of analyte --> Takes to long to process)! 

1. Salt content (Ionic content) goes up, Resistance goes down, current goes up to maintain and generate more heat.

2. Use constant  current to solve the problem. 

1. Horizontal : Old , cheap

2. Vertical: Closed system, good. No evaporation, No wick effect. 

1. Paper

2. Cellulose Acetate

3. Agarose 

4. Polyacrylamide Gel

Types of Electrophoresis support Media 

Paper

Non-restricting Support (Separate by mass ratio)

2. May interact with proteins

3. High endo0osmosis (No current) 

Problem: Polymer make up of cellulose, change paper??  

Types of Electrophoresis support Media

Cellulose

2. Non restricting supoort

2. Low endo osmosis 

Ph ~8.6 (Neg charge) since cellulose -> negative moves up to Positive 

Types of Electrophoresis support Media

Agarose (Carbohydrate from seaweed. high temp, mono)

(0.5-0.8% solution)

High Conc: Size pore decrease

Low Conc: Size pore increase, bigger pores

1. Variable pore size within a limited range. 

2. Primary use with large MW (Restricting method) 

( How big is the pore depends on how much in the solution)

3. Easy to use

Types of Electrophoresis support Media

Polyacrylamide Gel (1-3% poly)

Not good to get >10%

1. Wide range of pore size

2. Molecular Seiving (effective pore size depends by % cross linker in the polyacrylamide gel) 

3. Easy to shift by pH, very low MW

4. 

1. Non-denaturing

2. Denaturing

3. Separates by MW 

1. Urea

2. Mercaptoethanol

3. SDS

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Proteins 

Sensitivity vs. Non-sensitivity: Detect small conc proteins => (Deal with molar absorbtivity). 

a. Ponceau S

b. Bromophenol Blue

c. Amido Black

d. Silver 

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Proteins 

Only stain Hb. Nothing else. Only want to look at Hb.

Selectively apply stain 

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Proteins 

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Proteins 

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Proteins 

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Smear is black. Pick up every single protein, can't detect specific. Two types:

1. Sudan Black

2. Oil Red "O"

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Glycoproteins

Periodic Acid Schiff (PAS) - Specific dye applied.

Proteins glycosolated (Extracellular, gleucosamine, big carbohydrates) -> Apply specific dyes. 

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Enzymes

Enzymatic action with colored products.

Iso enzyme: Catalyze the same reaction. Apply the same concept. 

Put substrate to specific enzymes, warm the product to get the visual color --> Know the activity of that enzyme! Very important! 

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Non-Protein Compounds

Nucleic Acids

Separate DNA based on portion of DNA. Two types:

1. Silver (For big ones)

2. Ethidium Bromide (Binds to RNA and DNA.) Fluorescne dye apply to a gel to separate.

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Non-Protein Compounds

Amino Acids/Ninhydrin

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Non-Protein Compounds

Carbohydrates <-- (AgNO3)

Selective Dye

Ways to Locate the analyte after electrophoresis separation

Stains or Dyes 

Non-Protein Compounds

Lipids < -- Vaporized Iodine

Ways to Locate the analyte after electrophoresis separation

Autoradiography, Isotopic Constituents located by exposure on photographic film

Ways to Quantitate the analyte after electrophoresis separation

Densitometry

Determine the conc of total proteins in that specimen. 

STUDY PROCESS IN NOTES 

Ways to Quantitate the analyte after electrophoresis separation

Elution 

STUDY PROCESS IN NOTES 

Type of Disc Electrophoresis

Gradient Gel

1. Gel is cast with incremental dicreases in crosslinker. Looking at different size pores. Discontinuity Use for mixtures of protein, want to separate all. Go from top to bottom. Narrow the pore size to get good resolution. 

2. Separation by MW 

Type of Disc Electrophoresis

Stacker Gel

Discontinuity. Separate the band reflected the size of the sample that split in. Limit resolution. 

Restricting, very thin band 

+ sample concentraes at interface of resolving gel

+ Increase resolution 

Type of Disc Electrophoresis

Iso-electric Focusing

Discountinous BUFFER pH

Protein migrate stops at its isoelectric point (pH = 7) => Charge = Zero

Range of pH determined y the carrier ampholytes 

3. pH gradient established with mixture of ampholyte 

Type of Disc Electrophoresis

2D Electrophoresis

Complex mixture of protein, separated by electro. 
2. Sensation by one method in 1D, subsequent by second method in 2D.

3. High degree of resolution  

Types of Disc Electrophoresis 

Blotting

Isolate a separated band from the support media

2. Transfer to nitrocellulose

3. Electroblotting: Separate components are driven to the nitrocellulose by an electric current

+ High efficiency 

Southern Bloth ( For DNA)

Northern Bloth (For RNA)

Western Blot (For Protein - after electrophoresis)

Clinical Application of Electrophoresis

Serum Protein Electrophoresis

1. Cellulose acetate or agarose

2. Barbital buffer (pH = 8.6)

3. Amido Black 

Clinical Application of Electrophoresis

Hemoglobin Electrophoresis

* Reference method for differences in RBCs * 

1. Cellulose Acetate

2. Acid or Alkaline Citrate buffer, all stained with: 

3. Ponceau "S" 

Clinical Application of Electrophoresis

Lipoprotein

Variable support Media.

Test for Chloresterol

Always use Sudan Black!!! 

Clinical Application of Electrophoresis

Isoenzymes

1. Creatine Kinase

2. Lactate Dehydrogenase

3. Alkaline Phosphatatse,

used non-denatured, must as active enzyme. 
Use Agar.