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What is High Pressure Liquid Chromatography
there is still a column with beds, most commonly ion exchange beads. We take the column and incorporate it into a complex machine that makes the flow through the column a closed system
Explain the HPLC machine.
Cap on top and cap on bottom. They are connected to buffer reservoirs that are pumped in at high pressures.
Two buffers instead of one are used. You start with buffer A, then a mixture of A and B, and then end with buffer B
Final type of chromatography.
- one of the many denaturatnts we can use--> polyacrylamide gel electrophoresis
What are we doing with SDS-PAGE?
we are creating a stationer phase out of a molecule of acrylaminde. Exists as monomer-> cause linkage by cross-linking with methylenebisacrylamide --> gives jelly-like material
Structure of SDS-PAGE machine.
between two glass or plexiglass plates is the thin gel
At the top of the gel are indents (wells). All the area below it is the lane. Proteins are separated through the lane. The buffer is at the top and bottom
A current is sent through the gel, which grabs onto the proteins and pulls the proteins through the gel
__ help move the proteins (especially those with no charge.)
How does it work?
- Say we have a protein nicely folded--> add detergent
- --> causes hydrophobic tail to hide inside and the protein to eventually come apart
Denatured proteins with a negative SDS surrounding it. The amount of the negative charge corresponds to the length of the protein--> attracted to cathode
Also, have to deal with S-S bonds--> use beta-mercaptoethanol to reduce the bonds
We do what to the proteins and do what?
load the proteins into teh gel and turn on the power--> migration--> smaller molecules move faster
What happens when the gel is done running?
we see nothing. So, we stain it.
Drop gel in a buffer with the stain, Coomasie Blue, which interacts and binds to proteins. Bands are individual proteins
At the ends, what do you do?
you load molecular weight martkers (standard proteins)--> controls
We know everything about them, such as MW. We use them as our measurements.
What does SDS gel tell you?
What can this test tell you?
- If there was a band, the protein is there.
- If there is no band, there is no protein present.
It can be quantitative: different size bands= one had more protein than another. Two bands may indicate isomers
We ca do what for our proteins. How?
confirm the molecular weight of oru proteins
- Start from the well, measure to bottom of the well, called the front
- --> smallest molecule and moves the fastest
- Go to each band, start at the well, and measure down. This will give you the Rf value (how fast the object migrated)
What do you do with the Rf?
plot the Rf value against the log of the mass of the protein we measured (the standards)
What is special about SDS-PAGE?
it sits on the border of isolation and characterization
--> all the easy stuff as been done
What is bad about SDS-PAGE?
crude homogenate--> run on gel--> get all info
- Good because you get info
- bad because it denatures the protein
Last Chromatography technique?
- isoelectric focusing:relying on the isoelectric point
- Every protein has charged amino acid side chains and charges
- You can find a point where the net charge is zero= pI
What do you do with isoelectric focusing?
run a gel that determines this.
No SDS present. Instead are electrolytes (small molecules that contain different constituent groups) that, when you run the gel, you have a gel with a giant pH gradient (low pH to high pH)
this is the stationary phase
What is the stationary phase?
can be the gel or the paper
Let proteins migrate however they want.
__ migrates to __ and vice versa
Now that we can separate by size and charge, what can we do? Explain.
- Top: run the isoelectric focusing up top
- Vertical: take the results from the first and separate further by mass
Explain the result.
you have a fingerprint of the proteins--> every time you run it, it should give the same fingerprint
pattern is unique of the proteins that were present (can detect phenotypes if the pic varies)