Need Help with some Chemical Components For Those Smart Peop

[Chriseverclear]

Hi Guys,

I am trying to do a protein electrophoresis lab experiment.

SpoIIAA, Bacillus subtilis
SpoIIAB-enzyme

Objective: Phosphorylation of the protein changes its charge significantly (why?), but hardly affects the molecular weight of the protein (why?)

What does phosphorylation buffer do if i add them?

Do you guys know of any website where it tells me what each chemical components do? For example, Tris buffer, isopropanol....etc

thanks,
C.S

[canalon]

For components:
Tris is a buffer. Look for definition of this word)
Isopropanol is usally used to precipitate DNA since in presence of salt (usually Na Acetate) and in big enough concentration DNA is not soluble anymore.

Someone already made a list of the role of the most common components here

For your questions:
What is chemically speaking a phosphorylation? What do you add/remove? Is it charged or neutral? Is it heavy or light compared to a protein MW?

[Chriseverclear]

For components:
Tris is a buffer. Look for definition of this word)
Isopropanol is usally used to precipitate DNA since in presence of salt (usually Na Acetate) and in big enough concentration DNA is not soluble anymore.

Someone already made a list of the role of the most common components here

For your questions:
What is chemically speaking a phosphorylation? What do you add/remove? Is it charged or neutral? Is it heavy or light compared to a protein MW?

Here are the details.

Objective: Phosphorylation of the protein changes its charge significantly (why?0, but hardly affects the molecular weight of the protein (why?)

Step 1.
Pipette the following into a tube:
50 ul SpoIIAA (Stock solution is 3mg/ml)
12.5 ul SpoIIAB (Stock solution is 2mg/ml)
32.5 ul phosphorylation buffer
Pre-warm at 30 Celsius for 5 minutes.

Question1: Why do we need to add phoshorylation buffer? what does it do?I guess we pre-warm to facilitate the chemical reactions for 5 minutes?

Question2: Do you guys have any ideas what Native stop buffer does and also denaturing stop buffer? What do they do and what do they contain?

I hope this is much clearer.

thanks,
C.S[/u]

[canalon]

Impossible to say what a buffer contain unless you look into its composition, usually available on a leaflet with the tube (they can be useful) or on the manufacturer website if you have this kind of data. As for your 2 stop buffers, I would just say that one is denaturing (may contain Urea, disulfide bond reducers etc..) when the other one helps keep proteins in their native conformation.

As for your phosphorylation buffer it probably contains the reagents necesaary to have phosphorylation (so whatever salts needed for the phosphorylase, and the Phosphoryl-donor etc..) once again have a look at the comosition.