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BLD 414
Exam 3
105
Biology
Undergraduate 4
03/19/2013

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Term
Chemiluminescence
3 characteristics
Definition

NO enzyme, chem rxn. 

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

Term

Enzyme substrate on Chemiluminescne

 

 

Definition
Non-bounce fraction, depends on how much antigens (Low/high enzyme). Evaluated by substrate
Term
Substrate of Chemiluminescence
Definition
Enzyme as substrate has Product and light (photon)
Term
Bioluminescence and characteristics
Definition

Enzyme catalyzed. Product: Light

Greater specifictiy>Chemiluminescene

Has really low detection 

Term
Analyte of Bioluminescence and Reduce
Definition
Variable (ATP)
Reduce: Luciferase
Term
Types of Luminescents
Definition

Reaction Dependent

Derivitation 

Term
Reaction Dependent Luminescent
Definition

Light output proportional to analyte Conc

Luciferase assay for ATP 

Term
Derivitization
Definition

BInding of Luminescent substance to analyte

Required to separate free from bound

Enzyme immuno assay with perodidase labeled anibody 

Term
Instrument required for Luminescent assay
Definition
Put the filter to reduce interference. 
Put in multiple tues to measure, visualize.
More accurate result
Term
Goal of Enzyme Kinetic
Definition
Enzyme Conc. correlate with the activity of that enzyme
Term
Michalis -Menton Relationship
Definition

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 

Term
Enzyme kinetics rate
Definition
The rate is increase in Exponential, NOT linear
Term
Low Enzyme Substrate. What order?
Definition
First order Kinetic
Term
High Enzyme Substrate. What order?
Definition
Saturated -> Zero order. [Conc] is constant
Term
The Michalis Constant (Km)
Definition

Index of Enzyme Substrate Interaction

Km=1/2 Vmax

 

Term
Goal of Michalis Constant (Km)
Definition
Correlate activity to concentration. 
Monitor how much product formed at certain conc at low level substrate (No Linear Function)
Term

What does Km stand for?

 

Definition
The characteristics of that enzyme. 
Represents for the Realative Infinity of that substrate for that enzyme
Term
Km vs. Affinity
Definition

High Km: Low affinity for enzyme

Low Km: High affinity for enzyme

Term
How to determine the enzyme conc by rate of activity?
Definition
Correlate enzyme with activity product, only evaluated with linear phase of change. 
Product: Amount of absorbance
Term
How to determine linear of change for enzyme?
Definition
Fixed Period Method
Term
Fixed Period Method
Definition
Assume rxn rate is constant. Take two measurements T1 and T2. Calculate to determine for T1 and T2.
Term
How to measure T1?
Definition
Repeart measure 30 secs after everytime demonstrate it. Run at different enzyme conc. It takes Longer/lesser to saturated
Term
How to measure T2?
Definition
Start running the enzyme conc activity. Go linear beyond the point, get the conc -> Upper Limit of linearity. Take the degree of enzyme that's NOT CURVE. Bend over to produce the highest slope => Look at time for T2.
Term
Calculate the Enzyme
Definition
Term
Methods to Quantitative determine the enzyme conc by rate of activity
Definition

1. Kinetic Assay (Repeat max of rate)

2. Continuous Monitoring Method

3. Fixed Period Method 

Term
Kinetic Assay Method
Definition
Measurement at the max rate to represent the rate of activity
Term
Continous Monitoring Method
Definition

1. Lag phase

2. Zero order phase.

3. Bend over (Substrate depletion and Product inhibition)

 

Term
Osmolarity
Definition
Osmoles/Liter (Numbers of Species in solution)
Term
Osmolality
Definition
Osmoles/kg solvent (Dissociate 100%)
Term
Effects of Osmolality
Definition

Increase Osmolarity -> Decrease Boiling point

Increase Osmolarity -> Decrease Vapor Pressure

Term
Methods used to measure Osmolarity
Definition

1. Vapor Pressure

2. Freezing Point Depression Osometry 

Term
Vapor Pressure Osometry Components
Definition

1. Thermocouple

2. Sample Pad (have specimen on)

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

 

 

Term
Characteristic of Vapor pressure Osometry
Definition

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

2. Chamber temp = Dew Point temp.

Vapor Pressure related to dew point. 

Term
Vapor pressure vs. Dew Point
Definition

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.  

Term
Salt water vs. Boiling Point
Definition
Add Salt to water -> Increase boiling temp due to Increase Ionic content -> Vapor Pressure decreases.
Term
Osmolarity vs. Vapor pressure
Definition
High osmolarity -> Low heat of evaporation -> low vapor pressure (too few water with no ions inside)
Term
Vaporization
Definition
Refridge on
Term
Condensation
Definition
Below dew point, water precipitate, form water drops line up on surface.
Term
From Condensation to Dew Point
(Heat of Evaporation)
Definition
Refridge off. No pressure to keep the drop in liquid form, go back to gas (Liquid->gas)

Term
Dew Point
Definition
Monitor the temp for basic standard curve
Term
Vapor Pressure Osmometry Interferents, limit
Definition
Accurate required. Volatiles interfere, low precision.
Term
Freezing Point Depression Osmometry
Definition
Adding salt to increase Osmolarity, freezing point go wayyyyyy down.
Freezing point decrease -1.86*C for each osmole/kg solvent
Term
Component of Freezing Point Depression Osmometry
Definition

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.

 

Term

Freezing Point Depression Osmometry Characteristics

 

Definition

1. Sample in cell supercooled.

2. Rapid freeze seeded by agitation. 

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

Term
Antifreeze in Freezing point depression
Definition
(-6->-8*C). Pull the heat of the specimen very quick, rapidly. Super cool specimen
Term
Sitrring Wire
Definition
Detects super cool solution (Shake rapidly and produce multiple seeds). Initiate rapid crytalization.
Term
Super cooling Point
Definition
Temperature less than freezing pint, wait for crystal to catch up!
Term

How to quantitative the Osmolarity?

 

Definition
Series of standars. Microprocessor controlled calculations by using Alcohol.
Term
Basic principles of electrophoresis
Definition

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. 

Term
Goal of Electrophoresis
Definition
Separate the constituents of mixture. Want things to migrate fast!
Term
Cathode
Definition
Negative charge
Term
Anode
Definition
Positive Charge
Term
Components of Electrophoresis
Definition

1. Chambers

2. DC power 

3. Support Media.

4. Buffers 

Term

How does Buffers effect electrophoresis?

 

Definition
Buffers effect flow of bubble with direction. Relative pull to Anodes s Weak, very Weak Acids (Anode at 8.6 pH). Has big water hydration.
Term

What determines the rate of Electrophoresis migration?

 

Definition

1. Charge of molecules

2. Electric Field Strength

3. Friction (Viscosity, MW, Physcial Barriers)

Term
Physical barriers
Definition
Restricting/Non restricting? 
The only difference is change the ratio
Term
Restricting Barriers
Definition

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

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

Term
Non-restricting Barriers
Definition
Huge pores. The only differences is changing the ratio.
Term
Endo-osmosis
Definition
Charged support accumulates ions. Ionic increases. When Cathode moves to Anode, it generates heat.. The electrons fly through Bridge. If hot enough, proteins denature! The longer running - > The worse!
Term
Endo-Osmosis dissociate with Salt
Definition
salt is WA. Increases temp, increase disassociation of WA -> More dissociate -> Increase ionic, decrease resistance! ( current go up, more electrons get through), generate more heat!
Term
Electro Osmosis
Definition
Electric field, ion migrate to opposite poples bringing solvent. 
Impede forward migration of molecules, used as a mechanism of separation.
Term
Evaporation of Heat
Definition

1. Associated with current flow.

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

3. Might denature proteins. 

Term
Evaporation of Water
Definition

1. Increases the ionic strength of bffer

2. Wick effect 

Term
How does heat effect evaporation of water? Why is it related to Wick effect?
Definition

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,  

Term
How to minimize the effect of Heat?
Definition

1. Slow down evaporation.

2. Use constant current power source.

3. Refridgerate.

 

Term

How does Heat effect the current?

 

 

Definition

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. 

Term
Types of Electrophoresis Apparatus
Definition

1. Horizontal : Old , cheap

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

Term
Types of Electrophoresis support Media
Definition

1. Paper

2. Cellulose Acetate

3. Agarose 

4. Polyacrylamide Gel

Term

Types of Electrophoresis support Media 

 

Paper

Definition

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??  

Term

Types of Electrophoresis support Media

 

Cellulose

Definition

2. Non restricting supoort

2. Low endo osmosis 

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

Term

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

 

 

 

Definition

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

Term

Types of Electrophoresis support Media

 

Polyacrylamide Gel (1-3% poly)

Not good to get >10%

 

Definition

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. 

Term
Types of Polyacrylamide Gel electrophoresis
Definition

1. Non-denaturing

2. Denaturing

3. Separates by MW 

Term
Types of Buffers Denaturing Polyacrylamide Gel Electrophoresis
Definition

1. Urea

2. Mercaptoethanol

3. SDS

 

Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 


Proteins 


Definition

 

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

 

a. Ponceau S

b. Bromophenol Blue

c. Amido Black

d. Silver 

Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 


Proteins 


Ponceau S 
Definition

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

Selectively apply stain 

Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 


Proteins 


Bromophenol Blue
Definition
More sensitive > Ponceau S (Lower conc). Visualize few more porteins, see HB but also something else with LOWER CONC 
Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 


Proteins 


Amido Black
Definition
Relatively sensitive. Can see many Bands.
Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 


Proteins 


Silver
Definition
Use to isolate proteins with others. 
Most sensitive. Every proteins will come up, appear as smeal. Whole  
Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 

 

Lipoprotein
Definition

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

1. Sudan Black

2. Oil Red "O"

 

Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 

 

Glycoproteins

Definition

Periodic Acid Schiff (PAS) - Specific dye applied.

 

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

Term

 

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 

 

 

Enzymes

Definition

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! 

Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 

 

Non-Protein Compounds


Nucleic Acids

Definition

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.

 

Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 

 

Non-Protein Compounds


Amino Acids/Ninhydrin

 

Definition
Everything separate out, detects apply to hit Amino Acids specificly.
Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 

 

Non-Protein Compounds


Carbohydrates <-- (AgNO3)

Definition

 

Selective Dye

Term

Ways to Locate the analyte after electrophoresis separation

 

 

Stains or Dyes 

 

Non-Protein Compounds


Lipids < -- Vaporized Iodine

Definition
Selective Dyes
Term

Ways to Locate the analyte after electrophoresis separation

 

 

Autoradiography, Isotopic Constituents located by exposure on photographic film

Definition
Term

Ways to Quantitate the analyte after electrophoresis separation

 

 

Densitometry

Definition

Determine the conc of total proteins in that specimen. 

 

STUDY PROCESS IN NOTES 

Term

Ways to Quantitate the analyte after electrophoresis separation

 

 

Elution 

Definition



STUDY PROCESS IN NOTES 

Term

Type of Disc Electrophoresis

 

Gradient Gel

Definition

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 

 

Term

Type of Disc Electrophoresis

 

Stacker Gel

Definition

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 

Term

Type of Disc Electrophoresis

 

Iso-electric Focusing

Definition

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 

Term

Type of Disc Electrophoresis

 

2D Electrophoresis

Definition

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  

Term

Types of Disc Electrophoresis 

 

Blotting

Definition

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 

 

 

Term
Types of Blotting
Definition

Southern Bloth ( For DNA)

Northern Bloth (For RNA)

Western Blot (For Protein - after electrophoresis)

Term

Clinical Application of Electrophoresis

 

Serum Protein Electrophoresis

Definition

1. Cellulose acetate or agarose

2. Barbital buffer (pH = 8.6)

3. Amido Black 

Term

Clinical Application of Electrophoresis

 

Hemoglobin Electrophoresis

Definition

* Reference method for differences in RBCs * 

 

1. Cellulose Acetate

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

3. Ponceau "S" 

Term

Clinical Application of Electrophoresis

 

Lipoprotein

Definition

Variable support Media.

Test for Chloresterol

 

Always use Sudan Black!!! 

Term

Clinical Application of Electrophoresis

 

Isoenzymes

Definition

1. Creatine Kinase

2. Lactate Dehydrogenase

3. Alkaline Phosphatatse,

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

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