Question

Hi people can you please shed some light light on these questions for me:
1)what are the steps involved in the extraction of
- intracellular proteins.
- extracellular proteins.
2)describe the methods used in the extraction of
the intracellular proteins.

What is the difference between these two questions and what are the answers?

Thank you people.

Answer 1

@blues please help..

Answer 2

You get the main difference between the two right from the words: extracellular proteins are outside the cell while intracellular proteins are inside the cell.

Which, in conceptual terms, means to get at extracellular proteins you don't need to disrupt the cellular membrane (in fact, to avoid contaminating your sample with proteins from inside the cell, you should go to lengths to avoid disrupting the cellular membrane), whereas to get at intracellular proteins you will have to disrupt the cellular membrane.

There are a various different ways of getting the extracellular proteins out of their mediums. Some procedures are better for proteins in different kinds of tissue (i.e., plasma proteins in the blood vs. extra-cellular matrix proteins in solid tissues) as well on the type of protein itself and its specific way of attaching itself to membrane proteins, membranes with their various lipid and carbohydrate moieties, other extracellular proteins, etc.

Similarly, there are various ways of getting intracellular proteins out of their cells. Freeze thaw cycles, chemicals, etc all work, and all the various considerations discussed above also hold.

You should be very careful about distinguishing between just plain protein (as in all proteins) and proteins of interest. The steps above will give you all the proteins. But if you want just one (as typically you do) then you have to get up to further purification protocols - chromatography and HPLC, gels, etc - which give you the one protein you are interested in.

There are so many variations depending on cell type, protein type and desired end product that it is difficult to go into more than general detail on a question like this.

Answer 3

I should also add that your extraction scheme depends to great extent on what you want the proteins for.

For studies of protein structure, like NMR, GST pull downs which test for stable interactions and binding with other proteins, or activity assays, you want the protein in its folded form. With all those low energy bonds intact.

For studies of protein abundance or sequencing, you don't care if it comes out with all the structural integrity of a denatured noodle.

Answer 4

Hey @blues.. thanks loads.. actually I was asking for the difference between these two questions:
1) ")what are the steps involved in the extraction of
- intracellular proteins.
- extracellular proteins."
2)"describe the methods used in the extraction of
the intracellular proteins."

I know that it was a dumb question but actually I got those questions in class during the revision week and I was wondering if there was any difference between the steps involved in extraction of intracellular enzymes and the methods used in the extraction of the intracellular proteins.. That was basically what I was looking for.. I was just confused.. sorry..:/anyway, both comes to the same point.. steps are embedded in the methods if I may put it that way..

For industrial processes, centrifugation is the main method of separation-recovering the extracellular proteins from the spent fermentation medium. The main types of centrifuges are tubular bowl, chamber and disc. The purification is done by Adsorption chromatography, Affinity chromatography and Gel permeation chromatography.. Gas chromatography is used as well..

And to break open the cells to obtain intracellular proteins, mechanical methods such as sonification, pressure cells techniques could be used as well as the non mechanical method which involves the use of enzymes.. Once the cells are disrupted, centrifugation is carried out to separate the cell debris from the fermentation broth.. The supernatant is then extracted for purification.