While, in theory, using antibodies against Fc Receptors (FcRs) is the best way to eliminate unwanted signals mediated by FcR binding, you may not always have a well-optimized antibody pool against FcRs sitting right in front of you for your experiment. Furthermore, such a blocker may not be applicable when you have one of the FcRs, such as CD16, CD32 or CD64 to measure in your channel. In this blog, we share an "old school" way of blocking by just using a serum that is routinely available in most labs.
Protein phosphorylation is quintessential for specific pathways to function. This function can be influenced by internal or external factors. It is a reversible action with the involvement of kinases and phosphatases1. This activity is crucial in the cell cycle, apoptosis, and signal transduction pathways. If a protein is phosphorylated or dephosphorylated when it shouldn’t be, there can be severe disruptions to the cellular pathway. At times, the phosphorylation state can be the cause of a disease. This has been found in degenerative diseases, cancers, and various pathways involving the immune system2. For instance, kinase inhibitors have been successful in cancer treatments.
Enzymes control phosphorylation. Kinases function to add phosphate groups to proteins and phosphatases function to remove phosphates. Typically, phosphorylation occurs on serine, threonine, or tyrosine residues. This process can be very quick or it can take many hours. It results in a conformational change which either activates or inactivates protein activity.
In 2008, the Nobel Prize in Chemistry was awarded to Dr. Roger Y. Tsien, Dr. Osamu Shimomura, and Dr. Martin Chalfie for their discovery and development of the green fluorescent protein (GFP). Since then, fluorescent protein (FP) technology has made drastic advancements by many researchers. FP antibodies are used in multiple applications.
Green fluorescent protein (GFP) was first isolated by Dr. Osamu Shimomura in 1961. Since then, fluorescent protein (FP) technology has made drastic advancements by many researchers. Currently, FP technology is the most popular tool for visualizing target proteins. Dr. Shimomura, Martin Chalfie and Roger Y. Tsien won the 2008 Nobel Prize in Chemistry to mark their great achievements in life science research.
Living organisms on the earth synchronize their activity to a 24 hour light and dark cycle generated by the rotation of the earth. This biological rhythm is called the circadian rhythm.
Recently the molecular mechanism for the oscillation of the circadian rhythm has been elucidated and the approximately 24-hour-rhythm was found to be generated by a transcription-translation feedback-loop of clock genes expressed by almost all cells. In particular, BMAL1, CLOCK, PERs and CRYs play key roles in oscillation of the circadian rhythm and rhythmically regulate the expression of downstream genes (hereinafter referred to as clock-controlled genes [CCGs]).
So far, the expression analysis of CCGs is mainly performed by mRNA quantification, like qRT-PCR and in situ hybridization. However, it is necessary to analyze protein expression level, post-translational modifications like phosphorylation, and ubiquitination for understanding the molecular mechanism of circadian rhythm. Actually, phosphorylation and ubiquitination of PER and ubiquitination of CRY are well known.
Why are these protein expression analyses not well done? The answer is very simple: because there weren't any good antibodies in this market, so we developed antibodies against CCGs.
At first we developed antibodies against PER1/2, CRY1/2, BMAL1 and CLOCK, playing core feedback loop in circadian rhythm. BMAL1 and CLOCK form a heterodimer, which binds to the regulatory region (E-box) in per1/2 and cry1/2 to positively regulate transcription. PER and CRY proteins then form a complex, migrate to the nucleus, and negatively regulate the function of the BMAL1/CLOCK complex. CCGs with E-box elements are expressed and are regulated by the BMAL1/CLOCK complex, and various genes are expressed according to circadian rhythm.
We successfully developed antibodies against all of the core feedback loop members. Anti-PER1, PER2, BMAL1 and CLOCK can be used in Immunohistochemistry (IHC), and anti-BMAL1 and CLOCK can be used for Chromatin Immunoprecipitation (ChIP) assays as well. Please be careful to choose secondary antibodies because some of the products are derived from guinea pig!
Anti-PER2 (Mouse) pAb (Code# PM083) –Rabbit Ig (Aff.)
Anti-PER1(Mouse) pAb (Code# PM091) - Guinea Pig Ig (Aff.)
The path that an MBL antibody takes to go from development and manufacturing to the benchtop is intensive and rigorous. This meticulous attention to detail ensures that every vial a researcher uses maintains high quality and reliability. Here, we briefly describe the process.