Genetics Lab Final

Front 1

What ELISA stands for?

Back 1

Enzyme-linked immunosorbent assay

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Name of ELISA stands for three components

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Antibody (immuno)
Allows for specific detection of antigen of interest

Solid phase (sorbent)
Allows one to wash away all the material that is not specifically captured

Enzymatic amplification (assay)
Allows you to turn a little capture into a visible color change that can be quantified using an absorbance plate reader

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What ELISA is used for?

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Measure antibody levels (allergies, vaccines)

Detect viruses (hepatitis, HIV, Herpes, chicken pox)

Detect hormonal changes (pregnancy)

Detect circulatory inflammatory markers (cytokines)

Muscle damage (e.g. creatinine kinase) marker for heart attack.

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Enzymes with strong chromogenic substrates

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High molar extinction coefficient (i.e., strong color change)

Strong binding between enzyme and substrate (low KM)

Linear relationship between color intensity and [enzyme]

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Antibodies

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Specificity

Diversity – hypervariable region (1020 ~ 1026 combinations; human make ~ 108)

Affinity – range 105 < Kd < 109 M-1

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Sandwich ELISA

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1. Antibody coated well
2. Add antigen to be measured
3. Add enzyme conjugated antibody
4. add substrate and measure color

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Indirect ELISA

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1. Antigen coated well
2. Add specific antibody to be measured
3. Add enzyme conjugated secondary antibody
4. Add substrate and measure color

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ELISA plate

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96 well plate

Made of plastic on which protein can be adsorbed (bind) easily

Usually done overnight @ 4C (we don’t have the time)

Special buffer used that will not denature Ab and maximize binding

Blocking step ensures no empty spaces are left

Blocking reagent is often 10% BSA or Milk

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Standard Curve

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Serial dilutions of the Antigen (or protein mixture) being measured

Exact concentration is needed when determining the efficiency of an Antibody
A plot of concentration (mg/mL) is plotted against OD (optical density)

Not needed if your simply trying to determine if your Antigen is present within a protein mixture.

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Sensitivity of ELISA

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Typically defined as the lowest [Antigen] that can be detected above negative control

~2 S.D. above Mean Background Signal

Depending On Antibody Pair Used
Sensitivity Varies

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General Protocol of ELISA

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Dilute your unknown samples (5-fold serial dilution) w/ PBS

Ex.) Stock Solution of unknown will go in first sample well and then the next four wells will be 5-fold dilutions from there.

The capacity of the wells on the plate is ~300uL.
We will use 100uL for our experiments.

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ELISA steps

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Adsorb your Antigen (i.e. unknown protein samples) to plate

wash, wash, wash

Block unoccupied surface with Milk protein

wash, wash, wash

Incubate with Primary Antibody

wash, wash, wash

Incubate with Secondary Antibody (i.e. Enzyme-linked; HRP in this case)

wash, wash, wash

2o Ab Enz substrate = TMB (3,3',5,5' -tetramethybezidine) in buffer and H2O2.

Add 100uL of HCl and color should switch from blue  yellow (read at 450nm)

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Blocking

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Blocking Reagent 10% Milk
Add 200 L per well Blocking Buffer
Wait For 15 min at Room Temperature

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After Blocking

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Wash 3x with ELISA buffer [i.e. PBS/Tween (detergent)]
Add Samples
Samples are supernatants from bacterial culture or purified tissue sample (i.e. Na,K-ATPase)
Use 100 L of each Dilution

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Addition Substrate

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Add 100 L/well of TMB substrate

Incubate for ~10 mins, Avoid Formation of Excessively Bright Color (Spec will not be able to read)

Terminate Reaction by Adding 100 L/well 1.0 M HCl (color changes from blue to yellow)

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What goes in the blank during the plate set-up?

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Everything but Antigen!!
Then treat it exactly as the unknown samples

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Western Blot

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2-D gel blotted to nitrocellulose and specific proteins detected
with antibody.

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Quantitative Determination of Proteins

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No completely satisfactory single method to determine the concentration of protein in any given sample



Choice of method depends on the nature of the protein, the nature of the other components in the protein sample, desired speed, accuracy, and sensitivity of assay

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Methods Used for Protein Determination

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Biuret Test

Folin-Ciocalteu (Lowry) Assay

Bicinchoninic Acid (BCA) Assay

Dye-Binding (Bradford) Assay

Ultraviolet Absorbance

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Pros of Lowry Assay

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Sensitive over a wide range
Can be performed at room temperature
10-20 times more sensitive than UV detection
Can be performed in a microplate format

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Cons of Lowry Assay

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Many substances interfere with the assay
(Strong acids, ammonium sulfate )
Takes a considerable amount of time to perform
Assay is photosensitive, so illumination during the assay must be kept consistent for all samples
Color intensity varies with different proteins

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Bradford Assay

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CBBG primarily responds to Arg residues (8X more than other a.a. residues)

If you have an Arg-rich protein, need an Arg-rich standard.

showing increasing amounts of protein concentration

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Pros of Bradford Assay

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Fast and inexpensive
Highly specific for protein
Very sensitive [1-20 µg (micro assay); 2-20 mg (macro assay)]
Compatible with a wide range of substances
Extinction co-efficient for the dye-protein complex is stable over 10-orders of magnitude Dye-reagent complex stable for ~1 hour
Can be done in a microtiter plate.

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Cons of Bradford Assay

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Non-linear standard curve over wide ranges
Response to different proteins can vary widely, choice of standard is very important

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Immunodetection

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This procedure uses a primary antibody to detect a specific protein from the mixture blotted to the filter.

The antibody sticks to the protein. 

A secondary antibody is then added. It has a high affinity for the first (primary) antibody.

The secondary antibody has an enzyme, e.g.) HorseRadish Peroxidase (HRP), covalently attached.  

The enzyme produces a color by acting on a chromogen (e.g. diaminobenzidine [DAB]; 4-chloro-1-napthanol [4C1N])

Chromogens turn color as a result of enzyme activity. 

When the secondary antibody is present with 4C1N and H2O2 an oxidized brownish-purple color precipitates at the reaction sites and these bands are visualized.

Front 26

Differential Centrifugation

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This is the most common method of fractionating cells

Fractionation is the separation of the different organelles within the cell

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Differential Centrifugation Method

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1. Cut tissue in an ice-cold isotonic buffer. It is cold to stop enzyme reactions, isotonic to stop osmosis and a buffer to stop pH changes.
2. Grind tissue in a blender or hand-held homogenizer to break open cells.
3. Filter or low speed spin to remove insoluble tissue and unbroken cells
4. Centrifuge filtrate at low speeds (1000 x g for 10mins)
This pellets the nuclei as this is the densest organelle
5. Centrifuge at medium speeds (10,000 x g for 30 mins)
This pellets mitchondria which are the second densest organelle
6. Centrifuge at high speeds (100,000 x g for 30 mins)
This pellets ER, Golgi apparatus and other membrane fragments
7. Centrifuge at very high speeds (300,000 x g for 3hrs)
This pellets ribosomes

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Investigating Cell Function

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Differential Centrifugation allows us to look at each organelle within the cell

We can look at the individual organelles and study them in detail

This helps to determine each organelles function within the cell

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Centrifugation theory and practice

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Differential centrifugation

Density gradient

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Two types of rotor

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1 In the swinging-bucket rotor, at rest, the tube and bucket are vertical and the meniscus of the liquid is at 90° to the earth’s vertical centrifugal field.
2 During acceleration of the rotor the bucket, tube and meniscus reorient through 90° in the spinning rotor’s radial centrifugal field.
3 In the fixed-angle rotor only the meniscus is free to reorient – one of the reasons why open-topped tubes in particular must not be filled beyond the recommended level in a fixed-angle rotor, if spillage is to be avoided.

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Geometry of rotors

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The g-force or relative centrifugal force (RCF) in a rotor tube increases linearly with the radius, so the geometry of the tube with respect to the axis of rotation is important in determining the suitability of a rotor for a particular particle separation.
1 The rotor (or tube) of the swinging-bucket rotor (a) is routinely described by three parameters, the rmin, rav and rmax (the distance from the axis of rotation to the top, midpoint and bottom of the tube) and the RCF at each point is described as gmin, gav and gmax.
2 In a fixed angle rotor the value of rmin, rav and rmax (and the corresponding RCFs) is modulated by the angle of the tube to the vertical; the difference between rmin and rmax being greater in a rotor whose tubes are held at a wide angle to the vertical (b) than in one with a narrow angle (c).
3 The sedimentation path length of the rotor (or tube) is rmax – rmin. For tubes of equal volume and dimensions, the sedimentation path length is longest for a swinging-bucket rotor and shortest for a narrow-angle fixed-angle rotor.

Front 32

Calculating the RCF and rpm (Q)

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When calculating the rpm required to generate a certain RCF described in a published paper, be aware that the previous standard practice of always giving the RCF as the gav is no longer universally adhered to. Sometimes the RCF is quoted as the gmax, often no indication is given whether it is quoted as the gav or gmax.

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Differential Centrifugation

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Density of liquid is uniform
Density of liquid << Density of particles
Viscosity of the liquid is low
Consequence - Rate of particle sedimentation depends mainly on its size and the applied g-force.

Differential centrifugation is the simplest of the techniques used to resolve different types of biological particle and it is best illustrated by considering how it is applied to the partial purification of organelles from a tissue homogenate.

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Subsequent rounds of centrifugation and decantation

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In subsequent rounds of centrifugation and decantation, the RCF or the RCF and the centrifugation time are increased in order to pellet smaller and smaller particles. Each pellet is resuspended in the homogenization medium for analysis.

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Expected Contents of Differential Centrifugation

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800g pellet: Cellular Debris and unbroken cells

3000g pellet: Nuclei

15,000g pellet: Mitochondria, lysosomes, peroxisomes

Supernatant 2: cytoplasmic proteins, microsomes

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Large difference in the mean values of the pellets suggest...?

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contamination of the 1000g pellet with lysosomes and peroxisomes would be unlikely or that the 15,000g pellet should contain essentially no microsomes. But there is in fact considerable cross-contamination of every pellet, with either smaller or larger particles, or both smaller and larger particles.

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The problem of poor recovery and resolution

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associated with the movement of particles through the suspending medium is less pronounced as the sedimentation path length of the rotor is reduced. Low-angle fixed-angle rotors will therefore provide better separations than swinging-bucket rotors. The downside of using low-angle fixed-angle rotors is that the pellets are less compact. Because the centrifugal field is radial, only in swinging-bucket rotors is the pellet located at the bottom of the tube; as the tube angle gets less so the pellet gets displaced further up the wall of the tube.

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To improve the resolution of particles in an homogenate

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the various differential centrifugation fractions may be further fractionated in a density gradient. There are three basic formats: (i) a density barrier comprises just two layers, one of which is the sample; (ii) discontinuous gradients usually comprise 3-5 layers of different density, but can be as many 12 and (iii) continuous gradients in which the density increases smoothly (but not necessarily in a linear fashion) from top to bottom rather than step-wise as in (i) and (ii).

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How does a gradient separate different particles?

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Least Dense on top, most dense on bottom.

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buoyant density, equilibrium density or isopycnic banding

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magenta particles being much smaller take a much longer time to reach their final banding position

The problem for the magenta particles is that they have to travel through the banded red and green particles which may cause loss of resolution. The final equilibrium position is not dependent on the radial thickness of the sample layer, so long as all the particles are given sufficient time to reach their banding density.

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Summary of cell fractionation

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A particle will sediment through a solution if particle density > solution density

If particle density < solution density, particle will float through solution

When particle density = solution density the particle stop sedimenting or floating

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Resolution of Density Gradients

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Density barriers or discontinuous gradients, because of their very nature, tend to produce sharp bands of material at interfaces or at the meniscus or as a pellet, but continuous gradients are likely to provide the highest resolution, even though the bands will be broader (due to particle heterogeneity) and harvesting of the banded material may be less easy. Density barriers can be used to isolate the least dense particle (I) or the most dense particle as a pellet (II).

Front 43

Increase in substrate concentration

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observe change in enzyme activity