Experimental Methods Flashcards
What does Ion exchange chromatography exploit?
Differences in the sign and magnitude of the net electric charge of proteins at a given pH.
What is a column matrix in anion exchange chromatography?
A synthetic polymer containing bound charged groups.
If a column matrix has bound anionic groups, what is it called? What if it has cationic groups?
Cation exchangers
Anion exchangers
In ion exchange chromatography, what two factors affect the affinity of each protein for the charged group?
- pH (this determines the ionization state of the molecule)
- concentration of competing free salt ions in the surrounding solution.
Describe how cation-exchange chromatography works.
The solid matrix has negatively charged groups. In the mobile phase, proteins with a net positive charge migrate through the matrix more slowly than those with a negative charge, since the interaction of the positive/neg charges slows down positive proteins.
In size exclusion chromatography, what kind of proteins come out of the column first?
This purification process separates proteins based on size. In this process, large proteins emerge sooner than small ones.
How does size exclusion chromatography work?
The solid phase consists of cross-linked polymer beads with engineered pores or cavities or cavities of a particular size. Larger proteins cannot enter the cavities, so they take a short and rapid path through the column, moving around the beads. Smaller proteins get trapped by the cavities and are slowed on their decent.
What is affinity chromatography?
A form of chromatography based on binding affinity.
How does affinity chromatography work?
The beads in the column have a covalently attached chemical group known as a ligand. When a protein mixture is added to the column, any protein with an affinity for that ligand has its pathway through the matrix stopped, since it binds to the ligand. Once all of the protein that has no affinity is out of the column, you free your protein of interest is eluted using a solution with a high concentration of salt or free ligand.
Describe an SDS Page Gel
An electrophoretic method that is commonly employed for the estimation of purity and molecular weight. SDS (sodium dodecyl sulfate) binds to most proteins in amounts mostly proportional to the molecular weight of the protein.
- The bound SDS contributes a large net negative charge, rendering the intrinsic charge of the protein insignificant and conferring a similar charge-to-mass ratio.
- Electrophoresis in the presence of SDS therefore separates proteins almost exclusively on then basis of mass, with smaller proteins migrating more rapidly.
- After electrophoresis, the proteins are visualized by adding a dye that only binds to the proteins, not the gel.
What is isoelectric focusing used for?
A procedure used to determine the pI of a protein.
How does isoelectric focusing work?
- A pH gradient is established by allowing a mixture of low molecular weight organic acids and bases to distribute themselves in an electric field generated across the gel.
- When a protein mixture is applied, each protein migrates until it reaches the pH that matches its pI.
- Proteins with different pI’s are thus distributed differently throughout the gel.
What is an Edman degradation used for? What is a bonus of using this method?
Purifying protein by sequentially removing one residue at a time from the amino end of a peptide.
Useful because it does not damage the protein
How does an Edman degradation work?
Edman thought of a way of removing only one residue at a time, which did not damage the overall sequencing.
- This was done by adding Phenyl isothiocyanate, which creates a phenylthiocarbamoyl derivative with the N-terminal.
- The N-terminal is then cleaved under less harsh acidic conditions, creating a cyclic compound of phenylthiohydantoin PTH-amino acid.
- This does not damage the protein and leaves two constituents of the peptide. This method can be repeated for the rest of the residues, separating one residue at a time.
What is Sanger Sequencing used for?
Sanger sequencing, also known as the “chain termination method”, is a method for determining the nucleotide sequence of DNA.
How does Sanger Sequencing work?
There are three main steps for Sanger Sequencing:
1) DNA Sequence For Chain Termination PCR:
- The DNA sequence of interest is used as a template for a special type of PCR called chain-termination PCR. Chain-termination PCR works just like standard PCR, but with one major difference: the addition of modified nucleotides (dNTPs) called dideoxyribonucleotides (ddNTPs)
2) Size Separation by Gel Electrophoresis:
- The chain-terminated oligonucleotides are separated by size via gel electrophoresis. In gel electrophoresis, DNA samples are loaded into one end of a gel matrix, and an electric current is applied; DNA is negatively charged, so the oligonucleotides will be pulled toward the positive electrode on the opposite side of the gel. Because all DNA fragments have the same charge per unit of mass, the speed at which the oligonucleotides move will be determined only by size.
3) Gel Analysis & Determination of DNA Sequence:
-In manual Sanger sequencing, the user reads all four lanes of the gel at once, moving bottom to top, using the lane to determine the identity of the terminal ddNTP for each band. For example, if the bottom band is found in the column corresponding to ddGTP, then the original sequence has a guanine (G) base.
Describe the steps of a Western Blot
1) After prepping your sample, you load it into a gel electrophoresis to separate the proteins by size. SDS is also commonly used.
2) After running the gel electrophoresis, the proteins are then transferred to a solid support membrane by using voltage.
3) Detection of the correct protein is confirmed using antibodies.
- Once the transfer is complete, the membrane carries all of the protein bands originally on the gel. Next, the membrane goes through a treatment called blocking, which prevents any nonspecific reactions from occurring.
- The membrane is then incubated with an antibody called the primary antibody, which specifically binds to the protein of interest. Following incubation, any unbound primary antibody is washed away, and the membrane is incubated yet again, but this time with a secondary antibody that specifically recognizes and binds to the primary antibody. - The secondary antibody is linked to a reporter enzyme that produces color or light, which allows it to be easily detected and imaged.
What is a Native Gel Electrophoresis?
A gel electrophoresis that separates proteins based on their native charges, shapes, and sizes. Basically the same as an SDS PAGE except you don’t add the SDS.
How does an ELISA Work?
When performing ELISAs, antigen-antibody complexes are immobilized to a solid surface. An enzyme is covalently attached to one of the molecules in the complex, and the subsequent addition of an enzyme-specific substrate results in a colored reaction product.
Give the four steps to running an ELISA
1) Coating/capture—direct or indirect immobilization of antigens to the surface of polystyrene microplate wells.
2) Plate blocking—addition of irrelevant protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells.
3) Probing/detection—incubation with antigen-specific antibodies that affinity-bind to the antigens.
4) Signal measurement—detection of the signal generated via the direct or secondary tag on the specific antibody.
