[Week 7: Mar. 17-21] Western Blots

Proteins, proteins everywhere!

I've been learning how to do Western blots, also called protein immunoblots, a two-day process use to detect the presence of a protein in a sample. 

From the previous post, we have the samples listed in decreasing density: EGG, PELLET, and SUPE for each strain, so let's start off on what to do next: Western blotting.

Western Blotting 

We used Novex® Tris-Glycerine Express Protein Gel Electrophoresis Kits! The gels themselves are sticky, as I learned after getting them all over my gloves.

1. The first step is gel electrophoresis so we can separate the proteins by size with an electric current and have all the protein of interest in one place so it's easier to detect. We have pre-made gels so we don't have to pour our own. Don't forget to pipet a ladder into a well on each gel! 
2.  Then the proteins are transferred onto nitrocellulose, a kind of blotting paper that proteins stick to so your proteins will keep their pattern from gel eletrophoresis.
3. Afterwards the nitrocellulose is incubated in a buffer with genetic proteins (we used some from milk) so the antibodies won't get stuck all over the nitrocellulose that's sticky to proteins and mess with detecting our protein of interest. 
4. Next, we add our primary antibody. Remember how our strains of interest were created to have GFP or mCherry in their genome? The strains produce the protein of interest and the GFP or mCherry, linked together by several amino acids. The primary antibodies here are anti-GFP and anti-mCherry so they can tag the GFP or mCherry attached to the protein of interest. You might be wondering why the antibodies aren't anti-[protein of interest]. My understanding that it's easier and cheaper to make anti-GFP or anti-mCherry rather than to make an antibody with an antigen-binding site specific that has to be customized to the antigen associated with the protein of interest. Using the primary antibody lets us tag where our protein is.
5. After incubating for usually overnight, we then wash off our primary antibody and add our secondary antibody which is anti-primary antibody. The second antibody has horseradish peroxidase of another kind of protein or dye that we can use to see our sample later.
   
   You may be thinking: why two rounds of antibodies?
             Primary Antibody: Tags where our protein is (cannot be seen).
             Secondary Antibody: Helps us see where our protein is, how large it is compared                                                 to other proteins, etc..
   
6. Afterwards, the nitrocellulose blotting paper the protein are on can be visualized with a special method such as autoradiography and the intensities of the protein bands can be quantified. 

That's been my week, along with prepping for some other things, so I've been pretty busy. Hope you guys are doing well!

Reference:

• Davidson College, Department of Biology. (2001). Western Blot Procedure. Retrieved from Davidson College, Department of Biology website: http://www.bio.davidson.edu/genomics/method/Westernblot.html