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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

1

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

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

What can you infer when you see bands on a protein gel?

Quaternary structure

Disulfide bonds

Some subunits bigger than others