The basic function of these techniques is as follows:
A [SNOW] Blot helps find a specific section of [DROP] within a sample.
Ex: A Southern Blot helps find a specific section of DNA within a sample…
…and so on.
Let’s keep it general for now. What even is “blotting”? Every blotting procedure has four steps. These four steps are modified for the macromolecule type, but they are essentially functionally the same:
Use some form of gel-based separation to split the sample into sections based on molecular size.
Transfer these stratified molecules to a special surface. (This is the official “blotting” step.)
Use some form of chemical detection specific to the thing you want to isolate within the sample.
Visualize the results.
Let’s get into the specifics.
Southern Blotting
You have a sample of DNA and you want to isolate a section of DNA with a specific sequence. You cut it up using restriction enzymes to make it easier to handle on a gel - you need nice, small sections so things don’t get tangled later.
Use an agarose gel (which has relatively wider pores to accommodate larger nucleic acid pieces) to separate the solution of enzyme-processed DNA fragments. Larger fragments will be retarded by the gel (think of it like their greater size making it more friction-y) and will thus be at the top. Smaller fragments will swim through to the bottom.
The gel is placed on some liquid. A membrane of nitrocellulose is stacked onto the gel. By capillary action (adhesive force of liquid to medium that helps water climb up paper, for example), the liquid climbs into the gel and brings the DNA pieces into the nitrocellulose membrane. A transfer has occurred.
A DNA probe (single-stranded), designed to be complementary to the DNA sequence to be isolated, is added into the solution. It specifically binds the sequence of interest.
Use autoradiography to visualize the labeled bands (everything else is invisible).
One important thing to note is that RNA is an intermediate in the DNA expression process. Northern blotting can thus, unlike Southern blotting, provide insight into the expression profile for a given section of genetic material, by detecting its complementary mRNA.
Western Blotting
This one is for protein. It’s a bit different from Southern/Northern in multiple ways, so let’s look at the steps.
Prior to the Big 4 Steps of Blotting, we must first make the proteins more manageable. Kind of like restriction digestion in the Southern. Except way different. We don’t want to break the proteins into peptides because we usually want to isolate and analyze full proteins to determine their expression profile. Peptides would be useless for this.
So instead of trypsinization or something, we use Sodium Dodecyl Sulfate (SDS). This is a detergent that unfolds proteins (denaturation) and coats them with negative charge, masking all intrinsic charges conferred by amino acid R groups. This will become important.
Sidenote: Look at the first ingredient in your shampoo. You’ll likely see Sodium Lauryl Sulfate, which is a synonym for SDS. I think it’s so funny how Pantene conditioner proudly says “No sulfates!” and literally the first ingredient in Pantene shampoo is a freaking sulfate! When I get rich I’m getting those expensive natural shampoos istg. I mean, nothing wrong with using sulfate shampoo, but it can be drying.
Here are the Big 4 steps for a Western:
Use a polyacrylamide gel to finely separate proteins by molecular weight (polyacrylamide has smaller pores). You can also use an agarose, but polyacrylamide is more common for Westerns. Larger proteins on top, smaller rush to the bottom. Something important to note: in Northern and Southern blots, DNA and RNA are already negatively charged, so they didn’t need any special coating to enable the “large on top, small on bottom” pattern. But proteins, with their variable R groups, need a little support. Recall that we masked charges using SDS. That was to help with this. The use of polyacrylamide gel coupled with the SDS step is called SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).
This gel can be blotted onto a nitrocellulose membrane, but a PVDF membrane is also an option. For Southerns and Northerns, PVDF can be used, but nitrocellulose is more common and convenient for those. PVDF membranes are cool for Westerns because they enable chemiluminescent detection (you’ll see why this matters soon). Blotting occurs using electric charge: we coated our proteins with negative charge, so a positive charge is applied to the recipient membrane to attract the proteins. This process is called electrophoretic transfer.
Before adding a specific indicator of the protein sequence of interest, we need to make sure the indicator won’t randomly bind the membrane. This is also a concern with Northern and Southern blots, and has the same solution, but no one really talks about blocking in those contexts, which is why I’m explaining it here. To prevent nonspecific binding to the membrane, we use milk or bovine serum albumin (kinda NPC proteins) which coat the membrane and kind of repel the indicators. Anyway, the indicators used for protein detection include primary and secondary antibodies. The primary antibody binds to the protein sequence of interest, and the secondary antibody binds to the primary antibody and glows during the visualization process. It’s recommended to use different species’ antibodies for the primary and secondary to prevent nonspecific binding to sample protein sequences.
Visualization consists of looking at it. It’s common practice to use a chemiluminescent secondary antibody so we just look at it.
