Biochem 14.4 Protein Quantitation Techniques

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Questions and Answers

What is the molar mass of a protein with a mass of 50 kDa?

50 g/mol

25 kg/mol

100 kg/mol

50 kg/mol (correct)

What is a major limitation when quantifying impure protein samples using absorbance measurements?

Absorbance measurements are always consistent.

All proteins have the same absorption spectrum.

Absorbance cannot be measured at all.

The linear relationship between absorbance and concentration is limited. (correct)

Which assay methods are commonly used for protein quantitation of impure samples?

Bradford, EIA, and Western blot

PCR, spectrophotometry, and flow cytometry

NMR, HPLC, and ELISA

BCA, Bradford, and Lowry assays (correct)

Why is it common practice to produce a new standard curve for each experiment?

Absorption coefficients may not be consistent across experiments. Signup and view all the answers

What does the standard curve help to correlate in protein quantitation?

Measured absorbance and mass concentration of the protein. Signup and view all the answers

At what absorbance range is it common practice to choose protein standard concentrations?

0.1 to 1 AU Signup and view all the answers

Which component contributes to the overlap in absorption spectra of proteins and DNA?

The presence of tryptophan in proteins. Signup and view all the answers

Why are A280 and A260 measurements primarily used for relatively pure samples?

Contaminating substances can interfere with the measurements. Signup and view all the answers

What is the primary method used to quantify DNA and RNA?

Measuring absorbance at 260 nm Signup and view all the answers

What is the main reason why absorbance measurements of proteins may not correspond accurately to the number of moles present?

Proteins can vary in size and tryptophan content. Signup and view all the answers

To convert mass concentration of a purified protein to molar concentration, what is required to be calculated?

The molecular mass in Da Signup and view all the answers

What potential inaccuracies may arise from using average absorption coefficients for protein quantification?

They can oversimplify the relationship between absorbance and concentration. Signup and view all the answers

Why is it common to use an average literature value for a protein's absorption coefficient?

For convenience when a specific coefficient is not available. Signup and view all the answers

What are the main molecular residues that influence a protein's absorbance at 280 nm?

Tryptophan, tyrosine, and cysteine Signup and view all the answers

What does a higher absorption at 280 nm indicate about a protein's structure?

Higher tryptophan content in the protein Signup and view all the answers

When is additional measures necessary to determine biomolecule concentration from sample absorbance?

When the sample has poor absorptivity at λmax. Signup and view all the answers

What is the original concentration of a protein if a 10-fold dilution gives an absorbance corresponding to 0.45 mg/mL?

4.5 mg/mL Signup and view all the answers

What is the significance of the Kd value in binding assays?

It describes the binding strength between a protein and ligand. Signup and view all the answers

In the equation $Kd = \frac{[P] [L]}{[PL]}$, what does the variable [P] represent?

Concentration of free unbound protein. Signup and view all the answers

What happens during a serial dilution if a sample is diluted multiple times?

The overall dilution factor is the same as the individual factors. Signup and view all the answers

What is the primary reason visible light cannot be used to visualize proteins in microscopy?

Visible light has a longer wavelength than protein structures. Signup and view all the answers

What condition must be met for the free ligand approximation to be valid?

The ligand concentration must be much higher than the total protein concentration. Signup and view all the answers

What technique is used in cryo-EM to prevent damage to proteins during imaging?

Rapid freezing of samples in ice Signup and view all the answers

Which equation expresses the fraction of protein bound by ligand $(θ)$?

$θ = \frac{[L]}{Kd + [L]}$ Signup and view all the answers

What is necessary to visualize a three-dimensional structure of a protein using cryo-EM?

Multiple images from different angles. Signup and view all the answers

Why is the absorbance measurement of a sample important in concentration calculations?

It is an indicator of the sample's protein concentration. Signup and view all the answers

What is a key aspect of X-ray crystallography in determining protein structures?

The formation of crystals from the protein-containing solution. Signup and view all the answers

What is the relationship between total ligand concentration [Ltot] and free ligand concentration [L]?

[Ltot] is the sum of [L] and [PL]. Signup and view all the answers

Why is it significant that cryo-EM uses electrons for imaging instead of visible light?

Electrons have shorter wavelengths that can resolve smaller structures. Signup and view all the answers

What impact does low temperature have on samples in cryo-EM?

It slows the damaging effects of the electron beam. Signup and view all the answers

What is a disadvantage of the two-dimensional images obtained in microscopy?

They cannot be used to reconstruct three-dimensional models. Signup and view all the answers

How does the charged nature of electrons benefit electron microscopy?

It helps focus the electron beams with precision. Signup and view all the answers

How do different secondary structure elements, such as a-helices and β-sheets, affect the absorbance spectrum?

They absorb right-handed and left-handed circularly polarized light to different extents. Signup and view all the answers

What does the CD spectrum of a protein primarily measure?

The difference in individual right- and left-handed absorbance spectra. Signup and view all the answers

In a protein composed of 50% a-helix, 40% β-sheet, and 10% random coil, how is the ellipticity value calculated for a specific wavelength?

0.5 times the a-helix value plus 0.4 times the β-sheet value plus 0.1 times the random coil value. Signup and view all the answers

What is a primary reason for determining the structure of proteins?

To identify structural features that can inform about amino acid roles in function. Signup and view all the answers

Which of the following methods is NOT commonly used for the experimental determination of high-resolution protein structures?

Gas chromatography Signup and view all the answers

What recent advancement has improved the prediction of novel protein structures?

Use of artificial intelligence models with experimental data. Signup and view all the answers

How are CD spectra of proteins typically represented?

As a linear combination of the individual spectra of secondary structural elements. Signup and view all the answers

Which phenomenon could cause changes in a protein's CD spectrum?

Ligand binding or denaturation. Signup and view all the answers

What advantage does two-dimensional NMR provide in the analysis of molecular interactions?

It enables identification of which peaks interact. Signup and view all the answers

In protein NMR, what element is primarily used to probe the peptide bond?

15N Signup and view all the answers

How does chemical shift data in 2D NMR assist in understanding ligand interactions?

It can track shifts in peak positions as ligand concentration changes. Signup and view all the answers

What indicates a change in protein conformation when observing NMR chemical shifts?

Shift of peak positions to different ppm values. Signup and view all the answers

What specialized form of NMR is required for determining tertiary and quaternary structures?

Through-space interaction NMR. Signup and view all the answers

What advancement has improved the accuracy of protein structure predictions?

Artificial intelligence. Signup and view all the answers

How has the collection of protein structural data over decades influenced protein modeling?

It has provided a robust library for comparing proteins. Signup and view all the answers

What type of interactions does heteronuclear NMR help probe?

Interactions between different types of elements. Signup and view all the answers

Study Notes

Introduction to Additional Techniques

Biochemistry utilizes various techniques to study biomolecules and living systems, including electrophoresis, blotting, and chromatography.
This lesson provides a general overview of commonly used techniques, focusing on exam-relevant methods.
Knowledge of underlying principles of the techniques is important for understanding passages and data interpretation.
Biochemistry research often relies on principles covered in general, organic chemistry, physics, and biology.

Dialysis

Dialysis is a protein purification method used to exchange the elution buffer with a more compatible one.
The sample is placed in a container with a porous membrane.
The pores of the membrane are large enough for small molecules (salt, water, small ligands) to pass through but small enough that the target protein cannot.
The other side of the membrane is exposed to a desired final buffer (dialysate).
Small molecules/ions diffuse out of the sample and into the dialysate; desired solutes diffuse into the sample.
Water moves across the membrane to equalize osmotic pressure.
The process continues until solutes reach diffusive equilibrium.
Repeated rounds of dialysis are often necessary for further purification.

Hemodialysis

Dialysis is clinically relevant in hemodialysis, used for patients with impaired kidney function.
Patient blood is passed through a dialysis machine where it is exposed to dialysate fluid across a porous membrane.
Wastes diffuse from blood to dialysate, and essential electrolytes and small molecules diffuse from dialysate to blood.
Cleaned blood is returned to the patient.

Biomolecule Quantitation

Quantifying biomolecule concentration is crucial for various downstream applications, like electrophoresis.
Biomolecule concentrations can be measured in molarity or mass-per-volume units.
UV-Vis (ultraviolet-visible) spectroscopy is a common method for quantifying biomolecules.
The absorbance of a sample is directly proportional to its concentration, as expressed in the Beer-Lambert law: A= εcl. (ε=absorptivity, c= concentration of sample, l= pathlength).
The amount of absorbance is a result of molecules absorbing at a specific wavelength.

Absorbance and Quantitation of Impure Samples

Various methods exist for quantifying proteins, nucleic acids, and other impure samples. (eg. Bradford, BCA, Lowry).
A standard curve is often constructed to reliably correlate measured absorbance with concentrations.
The standard curve is produced using samples with known concentrations of the target substance.
The measured absorbance of the sample is used to determine the concentration.
Using standards (e.g. purified proteins or DNA) absorbance data are compared to a standard curve.

Serial Dilutions

Repeated dilutions can yield a series of increasingly dilute samples.
Dilution factors are multiplied to determine the original concentration.
Accurate quantification, including dilutions is essential in many biochemical assays.

Binding Assays that Assume the Free Ligand Approximation

Binding assays determine the interaction between a protein and a ligand.
The binding interaction is characterized by the dissociation constant (Kd).
Kd = [P][L] / [PL]
The fraction of protein bound (θ) can be calculated: θ = [PL] / ([P] + [PL]) = [L] / (Kd + [L])
The approximation that [L] ≈ [L_tot] is frequently used when the ligand concentration is substantially higher than the total protein concentration.
UV-vis spectroscopy can be used to analyze binding interactions.
Changes in the spectrophotometric properties (eg. absorbance or emission) indicate alterations in the binding state.

Isothermal Titration Calorimetry (ITC)

ITC measures the thermodynamics of binding reactions by monitoring heat released during ligand addition.
The change in heat release is indicative of the enthalpy of binding.
ITC allows the determination of the interaction without the free ligand approximation.
Determining binding thermodynamic parameters (H, G, S).

Melting Temperature Assays

Methods for measuring melting temperature (Tm) are available for proteins, nucleic acids, or other biopolymers.
Tm describes the temperature at which 50% of the biopolymer is denatured.
Differential scanning calorimetry (DSC) can be used where the heat capacity is followed while increasing the temperature at a controlled rate.
Melting temperature is also determined through spectroscopic methods, such as fluorescence.

Fluorescence

Fluorescence is commonly used to determine protein or other biomolecule conformation.
It involves excitation (absorption of a photon) followed by emission (release of photon with longer wavelength).
Fluorescent molecules (fluorophores) can be used or genetically encoded tags like GFP.
Spectroscopic techniques can assess protein conformation changes (eg. folding), as monitored by fluorescence or CD measurements.

Circular dichroism (CD)

CD is a spectroscopic technique that studies secondary structure in proteins and other biomolecules.
It measures the difference in absorbance of left and right circularly polarized light.
Protein secondary structure elements (eg. α-helices & β-sheets) produce distinct CD spectra.

X-ray Crystallography

X-rays can be used for atomic resolution protein structure determination.
XRC relies on diffracted X-rays from regular arrays of repeating molecules in the protein crystal.
diffraction patterns provide information on the three-dimensional arrangement of atoms and bonds.

Nuclear Magnetic Resonance (NMR)

NMR is another method for analyzing biomolecular structure.
NMR involves using radiofrequency pulses on nuclei in a strong magnetic field to reveal structural details.
Spectroscopic tools allow determination of primary or higher order protein structures.
NMR methods allow assessment of chemical shift interactions, and to monitor ligand interactions and conformational changes during structural analysis.

Cryogenic Electron Microscopy (Cryo-EM)

Cryo-EM is a high-resolution microscopy technique used to determine the structure of biomolecules such as proteins.
It involves imaging samples that are flash-frozen in ice, allowing for imaging of the 3D structure.
Many images are captured (of various rotated samples).
Images are used to mathematically reconstruct the 3D structure, and protein models are constructed.

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Description

This quiz covers essential concepts in protein quantitation, focusing on the methods and limitations when dealing with impure samples. Key topics include the molar mass of proteins, absorbance measurements, and the importance of standard curves in experiments. Test your knowledge of the techniques commonly used in biochemistry labs.