Login

Join for Free!
113964 members


Need Help with some Chemical Components For Those Smart Peop

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Need Help with some Chemical Components For Those Smart Peop

Postby Chriseverclear » Wed Apr 12, 2006 12:20 pm

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
Chriseverclear
Garter
Garter
 
Posts: 2
Joined: Wed Apr 12, 2006 12:14 pm

Postby canalon » Wed Apr 12, 2006 3:15 pm

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?
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
User avatar
canalon
Inland Taipan
Inland Taipan
 
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Protein Electrophoresis

Postby Chriseverclear » Wed Apr 12, 2006 4:14 pm

Canalon wrote: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]
Chriseverclear
Garter
Garter
 
Posts: 2
Joined: Wed Apr 12, 2006 12:14 pm


Postby canalon » Wed Apr 12, 2006 5:34 pm

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.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
User avatar
canalon
Inland Taipan
Inland Taipan
 
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada


Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 1 guest

cron