Class Notes (836,661)
Canada (509,870)
Biology (Sci) (2,472)
BIOL 300 (90)
Lecture

Lecture 26

5 Pages
108 Views
Unlock Document

Department
Biology (Sci)
Course
BIOL 300
Professor
Siegfried Hekimi
Semester
Fall

Description
th BIOL 300 November 7 2012 Lecture 26 Dr. Shock Biochemical analysis of signal transduction pathways were perhaps the first used experimentally. It was discovered that in addition to the minimal medium (the bare necessities of all cells, like amino acids, sugars, etc.), you also needed growth factors to to grow cells in culture • Scientists did this by grinding up cells, and adding the extract to the culture in addition to a minimal medium; this gave the cells everything they needed to grow • Sometimes, the cells would also change shapes or have different responses depending on the types of ligand in the extract • Even today, we don’t know the optimal conditions for tissue culture growth • A common additive is fetal bovine serum (FBS), which is serum of cow embryos, which helps tissue cultures grow nicely • This extract comes from cells in development because developing embryos have a lot of these signaling pathways turned on so there are a lot of ligands in the extract • Another type of cell that works well is the brain, which contains many secretory cell and signaling cells, which can be ground up and added What we are looking for in all of these cases of finding ligands are protein ligands (not small molecules like acetylcholine) • Because these proteins make up such a small proportion of the total protein in the cell, protein ligands are very hard to extract • They are almost all bound to receptor, and free ligand makes up an extremely small percentage of the total proteins • Biochemistry started by extracting abundant proteins like actin and myoglobin, which are much easier to extract How do you isolate a ligand biochemically? We can see here a ligand (Epo) in red binding to a receptor which is a heterodimer (blue and green). First, you need a tissue which contains high concentrations of the ligand you wish to extract • You can grind up different cell types to solubilize the proteins, and then feed them to different samples of tissue cultures to see how well they the cells grow You then need to have an activity test, which would be testing the sample extract on a tissue culture to see how well they let the tissue culture cells grow First, you need a tissue which contains high concentrations of the ligand you wish to extract • You can grind up different cell types to solubilize the proteins, and then feed them to different samples of tissue cultures to see how well they the cells grow You then need to have an activity test, which would be testing the sample extract on a tissue culture to see how well they let the tissue culture cells grow 1 th BIOL 300 November 7 2012 Lecture 26 Dr. Shock The first method used is ion-exchange chromatography: • First, you homogenize your tissue by grinding the cells up to create an extract which contains all the proteins in the cell • One of these proteins is the growth factor you want for your tissue culture • You can then separate proteins based on unique properties • One of these properties is charge given by the specific side-chains on your protein • The proteins are run through a gel containing positively or negatively charged beads depending on the protein you want to extract • With positive beads, the positive proteins will flow through the gel producing your flowthrough, while the negative proteins get stuck in the beads • The next step to get the proteins stuck in the column out is called elution • The desired proteins (in this case negative) would be in the elution • This step would cut down the pool of possible proteins by half Another important method is gel-filtration chromatography which allows to separate proteins by size (their other unique property) • The gel in the column contains pores of a certain size; proteins that are smaller than these pores get stuck and take longer than large proteins • The flowthrough would therefore have the largest proteins, and the elutions would have the smaller proteins • You would play around with the pore sizes in order to find the pore which best isolates your ligand. In reality, people often carry out both steps, first running through an ion-exchange chromatography to get only proteins of a certain charge, and then running that elution through a gel-filtration chromatography to further separate based on size You can then run the final run on an SDS gel to see the exact sizes of the proteins • Proteins are first denatures and made negative by SDS (to make them all have roughly the same charge), and then they are run on a gel • Smaller proteins will travel farther than 2 th BIOL 300 November 7 2012 Lecture 26 Dr. Shock
More Less

Related notes for BIOL 300

Log In


OR

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


OR

By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.


Submit