Gel Electrophoresis Flashcards
What is Gel Electrophoresis?
Technique used for seperating proteins or DNA based on size and charge using an electric field.
“electro” - EF
“Gel” - acrylamide (Proteins) or agarose (DNA)
Which is the Cathode and which is the Anode in Gel Electrophoresis?
The cathode is the negative end (reduction) and the anode is the positive end (oxidation). Proteins and DNA will migrate towards the anode which smaller proteins or peptide fragments migrating faster in the electric field.
Antiboodies are heterotetramers, held together extensively with disulfide bonds. When placed in Native conditions vs reducing conditions, what does the electrophoresis pattern look like for the intact IgG, Heavy chain, and Light chain
In Native, there is no reducing occuring and the heterotetramer remains intact so the “monomer” behaving group will not travel as far down the EF. However, in reducing conditions, the SDS is traveling seperately and will travel according to a charge/mass ratio.
What does the reducing conditions in gele electrophoresis (SDS) due to proteins?
Unfolds them, multiple subnits travel as monomers. However, if no reducing conditions, proteins are not unfolded.
In reducing conditions they are traveling according to charge and mass. While in Native conditions, they are charge/mass and molecular shape.
Describe the relation of pH and pKa in proteins.
If pH > pKa then the protein is negatively charged.
If pH < pKa then the protein is positively charged.
What is Isoelectric Focusing?
A gel is added that creates a stable pH gradient. (Large pores to prevent molecular weight seperation). Proteins are mixed and electric field is applied. The proteins either travel to the anode (positive) or cathode (negative) dependent on their isoelectric point. The protein on the L towards the cathode has a higher isoelectric point suggesting that it has more basic residues. Vice Versa.
In DNA sequencing, ddNTPs (ddG, ddA, ddT, ddC) are used. What are the purpose of these in comparison to normal dNTPs?
When DNA Polymerase is extending the DNA chain is incorporates a bond between the 3’ OH group and the alpha phosphate group. OH is a strong nucleophile. Once bond to ddNTP which substitutes an H for the OH there is no continuation of the strand.
Homodimer vs Heterodimer in Electrophoresis?
Homodimer is a protein complex that is made from the same protein subunits. So even if denatured completely for electrophoresis, there will only be one band because the individual subunits are the same. With hetero, you will see mulitple bands.
Differance between SDS-Page, Non-reducing SDS Page, and Native page.
Native - nothing is denatured, therefore you are looking at size, shape, and charge.
SDS - everything is denatured, so you are looking at individual subunits for everything and basing on mass/charge.
Non-reducing SDS - everything BUT disulfide bridges is denatured.
How does DNA sequencing work?
Template DNA (amplified by PCR) is mixed with DNA Polymerase and 5’ Oligo primer dNTP’s and a Buffer (solution that can resist pH change upon the addition of acidic or basic components). Then the DNA fragments and the the 3’ end a specific ddNTP is attached. After seperating all strands via electrophoresis, you can obtain the order of the DNA nucleotides.
Southern Blotting?
Used to identify specific sequences of DNA. Detects specific DNA sequence. Involves Agarose gel electrophoresis. Involves a capillary transfer. Uses nucleid acid probe which binds to DNA sequence and is XRayed.
What might suggest a size change on RNA in Northern Blotting results?
Alternative exon splicing, creating isoforms.
What is Northern Blotting?
Similar proceudre as Southern Blotting and is used for detecting specific seuqnces of RNA by hybridization with complementary DNA.
Detects specific RNA sequences.
Involves denaturing formaldehyde agarose gel.
Involves capillary transfer.
Uses cDNA probes.
IMPORANT: Used in gene expression analysis.
What is Western Blottin?
Used to identify specific amino-acid sequences in proteins.
Detects specific proteins by utilizing SDS page
Inolves an electric transfer
Uses primary and secondary antibodies
Used in disease diagnosis.
What is the Fab region of the antibody, and what is the Fc?
The Fab region is the region is the fragement-antigen binding region of the antibody where antigens can bind via their “epitopes” AA or sugars. There are two locations for antigens to bind to. The Fc region is the region that only has long heavy chains.
What is Gel Filtration (Size exclusion chromatography)
Agarose beads with defined pore sizes are used to separate molecules based upon differences in their molecular weights.
Works best with globular proteins, but smaller molecular weight is “includeded” or trapped first in the beads, while larger molecular weight is passed through “excluded” first (elution) because it cannot fit.
