Gel Electrophoresis Flashcards
How can DNA be cut up?
DNA can be cut up in many different ways.
Degraded
isolate DNA, and run that on a gel
pcr generates fragments of different lengths
DNA electrophoresis
Can separate strands of different sizes (can be RNA or other substances)
Smaller fragments move more quickly through the gel matrix
Resolution varies with percent agarose or percent acrylamide
Visualized with intercalating agents- Something like at ethidium bromide Sits between bases so you can see DNA. Dangerous because it can cause damage and mutations in live skin/DNA. Now just use things that fluoresce because its safer
Agarose gel
Agar comes from seaweed. Pour gel in a comb and it leaves wells. Put in a tray and put buffer with ions that run an electric current around the tray that is the same ionic concentration. Want sample to sink into the bottom of the well so the sample goes into the matrix of the gel
Why would you vary the percentage of agarose?
Higher percentage, smaller fragments can be separated
If you have pieces of DNA 400bp wanna use higher percentage agarose, same with acrylamide
Acrylamide gels
Have to be poured in between two glass plates. Air inhibits the polymerization of acrylamide. Buffer, put in acrylamide, add ionic salt that acts like a cross linker and helps make the gel matrix
DNA is negative, so it runs to the positive end(red). Depending how large the fragments are, the time will vary. Big ones are slow. Cross linked matrix- big things take longer to run through a gel
What can you determine from look at a gene sequence with a gel?
Looking at a gene sequence, humans are diploid so you can determine hetero/homo if there are one or two bands
What properties of a protein can be examined with gel electrophoresis?
Monomeric proteins: single subunit
Multimeric proteins: more than one subunit
Homodimers: dimeric proteins where both subunits are identical
Heterodimers: dimeric proteins where both subunits are different
How are proteins analyzed with gels?
SDS polyacrylamide electrophoresis
Weights dictate the size
The proteins run to the positive due to the SDS which gives each protein an equal mass to charge ratio, because proteins can have any charge
What are proteins treated with?
Treated first with SDS and then β‐mercaptoethanol
SDS maintains the denatured conformation of the proteins and coats the polypeptides with a negative charge, have an equivalent mass/charge ratio
Bigger proteins hold more SDS
β ‐mercaptoethanol reduces disulfide bonds , shows subunits
How are denatured proteins separated?
separated according to their MW on polyacrylamide gels
The higher the MW the slower it migrates on a gel
The number of bands corresponds to the number of subunits
The proteins are heated, denatured, then treated
What can you infer when you see bands on a protein gel?
Quaternary structure
Disulfide bonds
Some subunits bigger than others
