Monoclonal validated for Post-Translational Modification studies

Knowing the Human genome better has allowed major advancements in Personalised Medicine. Nowadays, we can know (if we want) the likelihood to develop a given disease and/or how we will react to different pharmacological treatments. Examples of this include diseases like breast cancer (for diagnosis or estimation of likelihood) and lung cancer (for response to treatment), to name just a few.

That said, our genotype does not have the last word. Research in the last couple of decades has shown the power of other regulatory mechanisms, that may enhance or diminish the effect that our genotype will have on our health. Starting from basic healthy life styles, to other more subtle mechanisms, our genotype defines us, but not completely. Above genetics, we have epigenetics… and everything at the protein level. This post will focus on Post-Translational Modifications (PTM), because, after all, it’s the proteins that are the final effectors of a given response to a treatment or to an environmental stimulus.

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How to get clear best-quality data in IP and WB protocols?

Immuno-precipitation & Western Blots often suffer from heavy  /light chain blotting, contamination, and ongoing interferences. This can prevent from obtaining biologically-relevant data in a given experimental model, especially when the target of interest has a size similar to the IgG molecules that may remain. Getting rid of these “artificial” bands can be tricky. In this post, you will discover the robustness of the TrueBlot® products when facing this issues.

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3 validated tools for ubiquitination studies

Elongation of ubiquitin chains, regardless of the linkage, can form polyubiquitin fibrils to initiate the Autophagy pathway. A deficiency of the autophagy causes cytotoxic accumulation of ubiquitin-positive aggregates leading thus  to neurodegenerative diseases (1). A new deubiquitinating enzyme, USP36, highly expressed in human breast and lung cancers, was identified to regulate c-Myc oncoprotein stability in nucleolus. (2) Also, Itch, an E3 HECT Ubiquitin ligase, inhibits MAPK p38α activation through ubiquitylation can be exploited therapeutically to prevent chronic skin inflammation (3). A recent publication by Yumimoto K. et al. determined that expression of FBXW7, the F-box Protein of SCF Ubiquitin Ligase, can suppress cancer metastasis in either non-cell-autonomous or cell-autonomous manner. (4)

Ub proteolysis pathway

Ub and F-box proteins proteolysis pathways.

 

Immunohistochemistry staining of formalin-fixed paraffin-embedded human lung using FBXW7 monoclonal antibody (3ug/ml) (Cat. No. 157H00055294-M02).

Immunohistochemistry staining of formalin-fixed paraffin-embedded human lung using FBXW7 monoclonal antibody (3ug/ml) (Cat. No. 157H00055294-M02).

In this work, researchers used the anti FBXW7 monoclonal antibody  to perform immuno-cyto-chemical staining on breast tissues.

Other tools to study ubiquitination include the use of Tandem Ubiquitin Binding Entities (TUBES) for the isolation and detection of polyubiquitylated proteins. TUBES can be used, in combination with Anti Linear Polyubiquitin antibody to investifgate Uquitin linkages on substrates.

References

  1. Morimoto, D., et.al. (2015). Nature communications. DOI: 10.1038/ncomms7116.
  2. Sun, X.-X., et.al. (2015). PNAS. DOI: 10.1073/pnas.1411713112.
  3. Theivanthiran, B., et.al. (2015). Science. DOI: 10.1126/scisignal.2005903.
  4. Yumimoto, K., et.al. (2015) JCI. DOI: 10.172/JCI78782.

If you are studying the role of ubiquitin and ubiquitination in your model, please leave your comment!

Cancer stem cells – friend or foe in the fight against cancer?

Today, following our series on Tumour Microenvironment (TME), and leading on from three recent publications, let’s discuss Cancer Stem Cells (CSCs).

CSCs are part of the TME, as they reside in niches where they can survive from immune surveillance, maintain plasticity and facilitate cancer metastasis (1). They can also circulate around the body. Which makes them so interesting. Their worse effect is that they can expand cancer to other organs in the body, thus helping metastasic. The good thing is that their early detection is a very powerful biomarker for cancer, allowing an early treatment. [Read more…]

DNA repair antibodies validated by MD Anderson

A series of publications (1, 2) in the past months has raised the never-ending debate on commercial antibodies and their validity in a given experiment. At tebu-bio, having sold antibodies for more than 40 years, we are well aware that not all antibodies fit all applications, and always strive to find the best antibody for a given experiment.

In collaboration with the MD Anderson Cancer Center, Rockland Inc. has developed and validated a highly specific toolkit to analyze PARP1 in a panel of control and siRNA knockdown cell lysates by multiple immunoassays. [Read more…]

Blood as a cells highway

MAB3838-98-12-1

IF on CTCs. HepG2 cells were stained with CD44-FITC labeled monoclonal antibody (Green). The cell nucleus were counterstained with DAPI (Blue) (Cat. No. 157MAB3838).

For years, we already knew that a lot of biomarkers are circulating in the blood (i.e. the secretome), as if it were a massive highway. Cells (apart from haematological cells, that is) also circulate.

There is much research now being focused on the role of several types of circulating cells in different diseases. Rapid advances in CRC research are pushing their utility to the in vitro diagnostic (IVD) arena, specifically in non-invasive diagnoses, companion diagnostics, and therapeutic monitoring. Up to date, several circulating rare cell (CRC) types have been described: Circulating Stem Cell (CSC), Circulating Endothelial Cell (CEC), Circulating Hematopoiectic Stem & Progenitor Cell (CHSPC), Circulating Mesenchymal Cell (CMC) and Circulating Tumour Cell (CTC).

Role of these CRCs still has to be elucidated, but there are some things we already know. For example, CSCs have a role in epithelial tumours & metastasis, as well as leukemia & lymphoma, together with CHPSCs and CTCs. CECs play a role in some tumour types. For cardiovascular, neurodegenerative and autoimmune diseases, CECs, together with CMSCs seem to be also involved.

Major challenges for the research on these CRCs is the optimisation of isolation methods, to make sure that we are indeed isolating CRCs and not other cell types. Current isolation methods depend on surface markers specific for these cell types, including EPCAM (1), CD44 (2), CD34 (3),  CD38 (4), CTNNB1 (5), etc.

Working on CRCs and their role in your disease? Leave your comments below!

 

 

References

1.- Zhao, M. et alAnal Chem. Oct 15, 2013; 85(20). doi: 10.1021/ac401985r.

2.- Qin, J. et al. Int J Clin Exp Pathol. 2014; 7(6): 3235–3244. PMCID: PMC4097256.

3.- Sho, E. et al. Arteriosclerosis, Thrombosis, and Vascular Biology, 2004; 24: 1916-1921.

4.- Ferrero, E. & Malavasi, F. J Leukoc Biol. 1999 Feb;65(2):151-61.

5.- Nowak, M. et al. Folia Histochem Cytobiol. 2007;45(3):233-8.

 

Invasion of influenza A

The influenza season in Europe is over… until next year. Influenza remains one of the most important public healthcare problems, especially in risk groups.

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Figure 1. Influenza A life cycle, indicating target molecules for which specific antibodies have been developed.Influenza virus can cause annual epidemics of approximately 500,000 deaths per year worlwide, which is facilitated by the high mutation rate of the viral genome and polymerase. The function interplay between hemagglutinin and neuraminidase glycoprotein characterises the pathogenic profile of each viral subtype (1).

The Influenza A virus (IAV), mimicked as misfolded protein aggregates, carries unanchored ubiquitin chains to exploit host cell’s aggresome processing machinery for efficient IAV entry and capsid dissociation (2).

For studies involving IAV, it is important that antibodies recognise the specific viral subtype, with high specificity, to avoid any cross-reactions with other subtypes that may not be relevant that season. [Read more…]

Notch and renal failure revisited

In our series of posts on different signaling pathways, let’s take a look today on Notch and its relevance in Acute Renal Failure (ARF).

A recent paper by Gupta et al. elucidated the role of the Notch pathway in kidney regeneration. This paper means an advance towards understanding potential therapeutic targeting of Notch signaling to enhance renal repair. Activation of the Notch pathway occurs following ARF. Pretreatment with the Notch ligand DLL4 enhanced recovery from ARF and represents a potential novel therapeutic option for regenerating the injured kidney.

100-401-407-Anti-Notch1-Antibody-1-IHC-4x3

Anti-Notch 1 (Cleaved N terminal) (Human specific) (RABBIT) Antibody (Cat. No. 039100-401-407).

However, compared to previous publications, as the authors mention in the paper, the use of different antibodies can affect the overall result of the experiment (as we all know!). In this specific case, Gupta et al. demonstrated increased expression of cleaved Notch1 and cleaved Notch2 as early as 1 h following reperfusion after 45 min of ischemia, and their findings are consistent with studies by Kobayashi et al. in a similar model of ARF with a few exceptions.

The paper by Kobayashi showed increased mRNA and protein expression of Delta-1, cleaved Notch2 only, while cleaved Notch1 was minimally detected under basal conditions or following injury. However, Gupta used the cleaved Notch-1 antibody from Rockland (see figure), and detected a robust signal for cleaved Notch1 with increased expression seen as early as 1 h following injury. These results were confirmed by immunohistochemistry using the Val1744 antibody. Therefore, both Notch1 and Notch2 are activated in the kidney following ARF.

Notch signaling has many roles, from neuronal function and development to the expansion of the hematopoietic stem cell compartment during bone development. Notch signaling pathways are a booming area of pharmacological research, due largely to the direct connection to human disease intervention.

Focus on apolipoproteins

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Role of apolipoproteins in different diseases.

Apolipoproteins, with amphipathic properties, form lipoprotein particles with phospholipids and transport hydrophobic lipids through lymphatic and blood circulations to designated peripheral tissues or organs for energy supply, biomolecule synthesis, or degradation. Off-balance of apolipoproteins has been implicated in numerous diseases such as SLE, myocardial infarction, Alzheimer’s, and diabetes. Apolipoproteins emerge as risk markers to pinpoint different diseases.

For Cardiovascular diseases, apoF and apoB seem to have a predominant role. [Read more…]

Biomarkers and shear stress

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Costaining of pAkt-1S473 (red) and PECAM-1 (green) after 30 min flow-adaptation and 5 min ortho- (D) or retrograde flow (E). Image was obtained using Cat. No. 039200-301-J34L for p-Akt staining.

Research nowadays aims at working on models as similar as possible to the real physiological status. This includes the modification of cell culture conditions, For example, one should perform cell culture under “real” oxygen levels (e.g. hypoxia, normoxia, physioxia). For circulating cells, shear stress is a key factor, as cells behave in a different way depending on whether they are cultured under static or dynamic conditions. [Read more…]