Protein

purica.on app tutorial: Ill do #10

of the complex mixture as an example

It will tell me something about the

protein

Where to start? It liked 50 C, which is

preEy warm, lets start with that

50 C for 20 min. More will precipitate if you heat it longer, but yours may also
precipitate. No harm in doing this over and over again!

Analysis tells us total protein (including contamina.ng protein), total enzyme describes
a total amount of enzyme by rela.ng it to how much total enzyma.c ac.vity there
is, giving us a reference to our total yield, and there is a cost with each step. Heat
treatment is cheap!

No.ce that total protein has dropped, but there is no drop in enzyme (units),
so we havent lost any of our enzyme, so we have 100% yield

Salt in/salt out is a good ini.al step

Uh oh! We dont want any of our enzyme precipita.ng, so 50% is too high.
You can hit cancel and you get a mulligan!

Well, we never did that 50% anymore! Keep lowering un.l you dont get any
enzyme precipita.on. How about 43%?

Good. 0.0% of enzyme, but we lose a third of total protein. Thats a lot of
contaminants that we have removed!

Were down to 389 mg of total protein, and we havent lost any of our
enzyme yet, so s.ll at 100% yield. Lets run a 2-dimensional gel to see all of
our contaminants and to nd out what our pI is for our protein.

Lots of proteins! Which is ours?

Hibng Blot tells us which one is specically ours. We have a pI of ~5.8, and a
MW of ~22 kDa

OK, we have a lot of op.ons for ion exchange. Lets try DEAE (posi.vely
charged, anion exchanger, no.ce the informa.on at the boEom, and it will
tell you all of this)

Well, we know the pI is ~5.8, so it should bind to a DEAE column at any pH

higher than that

Dierent condi.ons gives you dierent results. The blue curve is the A280. This is a
measurement of protein concentra.on. The red curve is enzyme ac.vity. This tells you
where your protein is. The pink line is an indica.on of the gradient.

pH 6, 0 to 0.5 M pH 7, 0 to 0.5 M pH 8, 0 to 0.5 M

pH 7, 0 to 1 M pH 7, 0 to 0.3 M pH 7, 0 to 0.2 M

Assay enzyme ac.vity gives you the

red curve. Lets run a 1D PAGE

Pick frac.ons in a range to check

S.ll a lot of proteins in with ours, but we got rid of a bunch. We can pool frac.ons
based on our gel and our ac.vity. No.ce that the ac.vity is more sensi.ve than the
gel. By the gel our protein is in 64-84, but by ac.vity it is in 60-86. No.ce that frac.on
72 looks pure, so we could just take that frac.on, but wed lose a LOT of our protein

We lost a small amount of units, but

thats OK. Its much cleaner!

Hydrophobic next! Phenyl-sepharose

should be good

Some proteins might not like the salt. If your enzyme isnt aected, go for it!
Free purica.on for you!

Hydrophobic gives us a terribly ugly

peak, but there is some resolu.on.

If we lower the amount of salt we start with, we can get our enzyme to bind
but the contaminant wont bind now at 1.8 M salt (instead of 2.0 M salt). This
is why stepwise gradients are so common in hydrophobic columns.

Looks good! We s.ll have most of our

protein.

A quick 1D PAGE shows one major impurity. Gel ltra.on is a good polishing
step! There are a lot of op.ons. There is some informa.on about these
columns if you scroll down further on this page

No.ce how dierent columns gives

dierent results

Purity is everything now! Lets throw away some protein to get rid of the
contamina.ng protein.

This is what wed like from you: Show us a single band on the gel, and give us the
EXACT condi.ons you used for each column (buer condi.ons, what column, what
frac.ons you collected, etc.) and you get bonus points for having the highest yield and
having the lowest cost. Im sure with some eort you can get more protein than I did
here and get it with lower cost.