Assay Readouts Lecture 3

Question 1

Factors affecting Absorbance

Answer 1

1. Impurities
2. Turbidity
3. Precipitates

Question 2

Absorbance Range

Answer 2

Most spectrometers display absorbance on the vertical axis, and the commonly observed range is from 0 (100% transmittance) to 2 (1% transmittance). The wavelength of maximum absorbance is a characteristic value, designated as λmax.

Question 3

Pros and Cons of Absorbance

Answer 3

1. Fast
2. Simple
3. No protein standard required
4. Assay does not consume the protein
5. Linear relationship

Question 4

Absorbance measurements of protein and nucleic acid

Answer 4

Protein is 280 and 205 nm

Nucleic acid is 260 nm

Question 5

Colorimetric Assays

Answer 5

A colorimetric assay is a quantitative analysis of solutions by estimating the color produced by the reaction of the sample with the reagent and comparing it with the colors produced by known standard solutions by using a colorimeter.

Question 6

Bradford Assay

Answer 6

1. Coomassie brilliant blue G250 when bound to protein (arginine, lysine and aromatic residues) causes an absorbance shift.
2. Cationic (unbound) form dye has an absorbance maxima at 465 nm
3. Anionic (bound) form shifts the absorbance maxima to 595 nm
4. The ratio of absorbance at 620 and 465 nm be used for the quantification of proteins

Question 7

Detection Limits of Bradford

Answer 7

1-20 micro assay

20-200 micro gram (macro assay)

Question 8

Advantages of Bradford Assay

Answer 8

1. Rapid
2. Inexpensive
3. Sensitive
4. Specific

Question 9

Disadvantages of Bradford Assay

Answer 9

1. Response to different proteins can vary widely and hence choice of standard is very important.
2. The high dependence of the assay on protein composition presents a major problem to the broad use of CB binding as a quantitative protein assay

Question 10

Considerations for Bradford Assay

Answer 10

Use glass or plastic cuvettes than quartz

Buy the prepared reagents instead of making one

Question 11

Factors Affecting ELISA

Answer 11

1. Affinity
2. Specificity
3. Blocking and washing
4. Choice of antibody
5. Capture ELISA - Ensure that capture and detection antibodies do not interfere with one another and can bind simultaneously

Question 12

Efficiency of the Chemiluminescence

Answer 12

Quantum yield

Question 13

Enhanced Chemiluminescence

Answer 13

Enhanced chemiluminescence is achieved by performing the oxidation of luminol by the HRP in the presence of chemical enhancers such as phenols, which in turn is mixed with a substrate, hydrogen peroxide (H2O2).

Light output is increased approximately 1000 fold and extending the time of light emission.

Question 14

Fluorophore

Answer 14

Fluorophore is a substance that absorbs light

Polyaromatic or heterocycles

Question 15

Fluorophores in microscopy

Answer 15

1. Chemical dyes - Need to be coupled to a macromolecule - Rhodamine; Phalloidin to F-actin
2. Fluorescent Proteins - need to be fused genetically to protein of interest - GFP
3. Biologically active ones - Bind to cellular/sub-cellular structures - DAPI

Question 16

Desired characteristics of Fluorophores

Answer 16

1. They should yield bright signals that are spectrally distinguishable from one another.
2. They should have minimal effects on the electrophoretic mobility of the DNA conjugate or the function of the protein.
3. Dyes should be compatible with available instrumentation.
4. Reactive forms of the dye must be available, in order to couple the dye to oligonucleotide primers or protein.

Question 17

Characteristic properties of fluorophores

Answer 17

1. Molar Extinction Coefficient
2. Quantum efficiency
3. Quantum yield

Question 18

Stokes Shift

Answer 18

Stokes shift - The difference in frequency or wavelength between the excitation and emission peak (hγex – hγem) is called the Stokes’ shift. This shift represents the energy dissipated as heat during the lifetime of the excited state before the fluorescent light is emitted

Question 19

Fluorescence Lifetime

Answer 19

The time delay between the absorbance and the emission

Fluorescence lifetimes are very short. Most fall within the range of hundreds of picoseconds to hundreds of nanoseconds.

Fluorescence lifetime of a molecule is very sensitive to its molecular environment, hence measurement of the fluorescence lifetime(s) reveals much about the state of the fluorophore.

Question 20

Major factors affecting fluorophore performance

Answer 20

1. Molar extinction coefficient
2. Quantum yield
3. Photostability

Question 21

Background fluorescence

Answer 21

1. Endogenous sample constitiuents
2. Unbound or nonspecifically bound probes
3. Crosstalk
4. Bleeding
5. Electronic noise

Question 22

Photobleaching influencers

Answer 22

1. Chemical reactivity
2. Intensity and wavelength
   3, intracellular chemical environment
3. Free oxygen radicals

Question 23

How to reduce photobleaching

Answer 23

1. Appropriate choice of fluorophore
2. Limit exposure time
3. Use of antifade agents

Question 24

Quenching

Answer 24

Reduces fluorescence intensity as a result of the presence of oxidizing agents or salts of heavy metals or halogen compounds

Quantum yield (reduced emission but not emission spectrum) is altered by interactions with other molecules.

Quenching sometimes results from the transfer of energy to other “acceptor molecules” close to the excited fluorophore – Resonance energy transfer

Question 25

Essential elements of fluorescence instrumentation

Answer 25

Excitation source Fluorophore Wavelength filters Detector