Fc blockers are needed for monocytes, macrophages, B cells and granulocytes. Since T cells normally do not express Fc receptors, some investigators do not use Fc blockers and instead use a dump channel (e.g. CD14/CD19) during flow cytometry analysis to exclude Fc-expressing populations.
The thymus shapes a host's T-cell repertoire so they do not respond to "self" antigens, yet are able to recognize "non-self" antigen challenges (from pathogens and tumors). T cell participation in these immune responses are dependent on interaction with peptide- and self-MHC. In other words, T cells only recognize the peptides derived from cancers or pathogens if they are embedded in the cleft of self host MHC grooves. Such peptides are processed and presented on the surface of antigen-presenting cells (MHC class I and II in dendritic cells, macrophages, and B cells) or all nucleated cells (MHC class I) allowing T cells to identify infected or cancer cells. Direct detection of such Ag-specific T cells and functional analysis is important for understanding disease immunopathogenesis and the process of drug development. MBL International has had tremendous success in advancing the methodologies to accurately measure antigen-specific T cells.
As described in previous blog posts, our IL-18 related products are used widely among researchers around the world. IL-18 plays an important role in the cytokine network. Abnormal values of IL-18 are seen in various disease areas such as allergy or autoimmune diseases.
In this blog, we discuss two diseases correlated with IL-18. Neither of them are allergy or autoimmune diseases, but very interesting nonetheless.
Interleukin-18 (IL-18) is a member of the IL-1 superfamily and synthesized as an inactive precursor that requires processing by caspase-1 to become an active cytokine. IL-18 plays a major role in the production of interferon-γ (IFN-γ) from T cells and natural killer cells. Refresh your memory by reviewing a couple of previous blog posts.
Interleukin-18 (IL-18) is a member of the IL-1 superfamily and synthesized as an inactive precursor requiring processing by caspase-1 into an active cytokine. IL-18 plays a major role in the production of interferon-γ (IFNγ) from T cells and natural killer cells. Refresh your memory by reviewing a couple of previous blog posts.
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.
Have you heard of the S100 protein family? S100 refers to proteins that are 100% soluble in saturated ammonium sulfate solution. They were first discovered as a major protein fraction (0.6% of total soluble proteins) isolated from bovine brain by B.W. Moore in 1965.
Mechanisms of Autophagy
The ubiquitin-conjugating protein p62/SQSTM1 is thought to be a scaffold protein that interacts with a variety of molecules involved in toll-like receptor (TLR) signaling, such as TRAF6, ERK, and aPKC. Recent evidence suggests that p62 binds directly to the autophagosome marker LC3 and is then selectively degraded by autophagy.
p62 contains multiple phosphorylation sites. Sequential phosphorylation of these sites regulates biological defense mechanisms such as selective autophagy and the Keap1-Nrf2 system. Therefore, the failure of the p62 pathway is associated with various diseases such as cancer and many neurodegenerative diseases.
To learn more about the mechanisms of p62, please refer to our whitepaper written by Drs. Masaaki Komatsu and Yoshinobu Ichimura, Niigata University. They are leading researchers in Autophagy, especially p62 and Keap1-Nrf2 interaction. Dr. Komatsu was recognized as one of the most highly cited researchers in Autophagy in 2014 and 2015 by Clarivate Analytics.
How do you detect p62 or phospho-p62 using antibodies? Western blotting? Immunohistochemistry? Quantification of biomarkers is essential for medical and diagnostic application. Selective autophagy and Keap1-Nrf2 are already being discussed for their use as a diagnostic as well as other medical applications.
MBL International has developed new sandwich ELISA kits that can semi-quantify total p62 and its phosphorylation level at Ser403 and Ser349. Phospho-p62 ELISA kits are the first kits to quantify these markers by ELISA in the world research market. It is a chance to get a new discovery in the Autophagy area!
|CY-7055: CycLex® Total p62 ELISA Kit|
|CY-7057: CycLex® Phospho-p62 Ser403 ELISA Kit|
|CY-7056: CycLex® Phospho-p62 Ser349 ELISA Kit|
These kits can help you to prepare cell lysate easily including cell lysis buffers. Phosphatase inhibitor solution is also included in the phosho-p62 kits. They can be used for human and mouse samples.
Total p62 and phospho-p62 levels in HeLa cells
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.)
MBL’s CoralHue fluorescent product series is composed of many proteins to make your life "brighter"! We offer expression vectors and cloning vectors. How do you know which vector to use? Learn more about how to choose which vector is right for you.