Jump to content

Recommended Posts

hi i m studing "Exploring Genomes and Proteomes in practice"

 

and its written that u can disrput the cell membrane either by

repeated freeze-thaw cycles, osmotic shock, grinding or

homogenization.

 

The resulting homogenate is fractionated (separated into individual fractions) by centrifugation, chromatography or electrophoresis.

 

 

so can someone explain it to me in brief

Link to post
Share on other sites

It sounds to me that you have to read up the basics of separation methods. I cannot (and also do not want to) give you an ad-hoc lecture here. Just some basic infos, for real understanding you should look them up in more detail in textbooks for bioanalytical techniques (check your local library).

 

In very short: electrophoresis is a technique to move charged molecules. For protein separation polyacrylamide gels are used that function as sieves. Larger proteins move slower through the gel than smaller ones, hence you can separate proteins according to their size that way. Of course you have to mask the endogenous charge of the protein first, but let's not confuse you too much.

 

For protein separation via chromatography normally HPLC (high performance/pressure liquid chromatography) is used. Essentially proteins are moving in a mobile phase through a column packed with the stationary phase. Depending on what the stationary phase consists of, the proteins are retained according to specific property (e.g. charge).

Link to post
Share on other sites
  • 2 months later...

latteral meanig of affinity is atraction. Afinity chromatography works on this principal. Sepration on the basis of positive ions or negative ions. Supose for the sepration of DNA we have to prepare the medium which contains positive ions because DNA is negatively charged. so that it binds with the medium and rest of the materials passes out.

Link to post
Share on other sites

Actually you can separate according to quite a variety of properties including, but not limited to size, charge and hydrophobicity.

Also depending on what you use as the mobile phase or how you derivatize your sample you can change the properties in such a way that allows separation of the substances in question.

Link to post
Share on other sites
  • 1 month later...

IIRC you can also use "tags" for specific proteins or nucleic acids during purification. There are several different ways to do it, but one common way would be to biotinylate (use of biotin as a tag) your molecule (protein, DNA, etc) of interest and then use avadin to select it from the rest of the cellular "stuff".

 

Another favored method will use anti-bodies. This is generally done for difficult to isolate or unstable proteins that don't do as well with normal methods of protein purification. It's generally quite expensive and labor intensive.

Link to post
Share on other sites

Yes, you can use tags, but this does not allow a separation and maybe subsequent identification of all the proteins in crude extract (as indicated in the op).

You can either capture only those that have been tagged genetically (e.g. using Hist-tags) or you would more or less randomly (in many techniques the lysine residue is targeted) label everything. This is normally not the goal if you want to analyse a crude extract.

Link to post
Share on other sites
Yes, you can use tags, but this does not allow a separation and maybe subsequent identification of all the proteins in crude extract (as indicated in the op).

You can either capture only those that have been tagged genetically (e.g. using Hist-tags) or you would more or less randomly (in many techniques the lysine residue is targeted) label everything. This is normally not the goal if you want to analyse a crude extract.

 

:doh:

 

Kind of missed the whole "proteome" part.

Link to post
Share on other sites

Create an account or sign in to comment

You need to be a member in order to leave a comment

Create an account

Sign up for a new account in our community. It's easy!

Register a new account

Sign in

Already have an account? Sign in here.

Sign In Now
×
×
  • Create New...

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.