Could you cover regulated vs constitutive promoters again?

Promoters are stretches of DNA where RNA polymerase can bind. There are many different promoter sequences, and it is the unique pattern of these sequences and regulatory regions (the up and downstream sequences that attract proteins that can bind DNA and modify RNA transcriptional activity through a variety of mechanisms) that control when a gene is transcribed and how many copies are transcribed. Constitutive promoters are “always on” – meaning that RNA polymerase is constantly binding to the promoter and transcribing. Regulated promoters have properties up or downstream that prevent (inducible) or block (repressible) transcription through any of a variety of molecular mechanisms.

Is there a way to alter protein generation and degradation rates?

Absolutely! This is actually a wonderful question for a biochemist, and I’ll point you towards literature in that field for more details. Briefly, protein synthesis, as defined by the time it takes from turning “on” transcription to the accumulation of a certain quantity of a protein, is influenced by transcription rate, mRNA stability, ribosomal binding site specificity, temperature, the presence of accessory folding proteins in the endoplasmic reticulum, and many other factors. Post-translational stability is impacted by ubiquitination and other post-translational modification that regulates targeting to the proteasome and proteasomal degradation rates.

If an antibody binds to a specific protein, why do you need to block it from the membrane? Shouldn’t it only bind to the protein?

An antibody is actually only specific to an “antigen” or an “epitope” – these are smaller regions of the larger 3D structure and can be very specific; we’ll cover it in the next lecture.

https://www.abcam.com/protocols/antigens

Could you point me to additional resources for antibodies and Western Blots?

There are a couple videos linked on the Week 10 page that might be helpful. As for reference-level additional resources, much like Addgene is to plasmids, there are several major commercial sources for antibodies and other protein biochemistry-related reagents. These companies benefit from customers understanding the technical details of what their products are and how they are made, so they have produced some very good resources where you can learn everything you’d ever want to know about the myriad protein biochemistry techniques… just keep in mind as you read these resources that they may be biased towards one company’s offerings.

Dako (now part of Agilent): “Immunohistochemical staining methods 6th edition” is a resource for histopathologists and, in my opinion, offers an ideal level of detail explaining what antibodies are and their practical use in the lab. The appendices cover in more detail what we touched on in class about primary antibodies, secondary antibodies, affining, reactivity etc… If you find yourself in a situation where you need to develop a staining protocol for any antibody, please take a peek at this reference first!  https://www.agilent.com/cs/library/technicaloverviews/public/08002_ihc_staining_methods.pdf

Abcam: makes antibodies and makes a concerted effort to track where and how they are used. The also publish really good protocols with plenty of background information. Abcam’s protocol section is usually my first stop when learning a new assay or to find a tried and true protocol for adaptation.  https://www.abcam.com/tag/western%20blot%20protocols

ThermoFisherScientific: is a huge life sciences reagents company. They have a user-friendly interface with bite-sized information about antibodies.   https://www.thermofisher.com/us/en/home/life-science/antibodies.html

Cell Signaling Technologies: makes very high-quality, diagnostic-grade antibodies. Here’s a link to their resources page – you can select your antibody and western blot area of interest. https://www.cellsignal.com/contents/resources/applications/resources-applications

Why is a secondary antibody needed in Western Blotting? Can the primary not be fluorescent?

The primary can be directly conjugated to a fluorophore, and for some very sensitive applications (ex: flow cytometry), the primary is usually fluorescent and no secondary antibody is used. One reason to use a secondary antibody is to increase the signal. One molecule of primary binds one molecule of antigen, but many molecules of secondary can bind to a single molecule of primary antibody. Sometimes, when the antigen abundance is very, very low, a tertiary antibody can be used on an additional signal amplification method like TSA (link).