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.

[Read more…]

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!

Measuring total ubiquitinated proteins in cell lysates – made easy!

To date, detecting changes in the ubiquitylation of specific substrate proteins in response to external stimuli, e.g. stress, cytokine exposure, drug candidate treatment, etc., has been a long, labor-intensive process involving immunoprecipitation followed by gel electrophoresis and Western blot analysis.  This method is low through-put, resource intensive, and only semi-quantitative at best.

ELISA like method to measure all ubiquitinated proteins in cell lysates

Ubiquant S results 1

Fig. 1.: Decrease in LDLR ubiquitylation following treatment of transfected cells with an IDOL inhibitor.

To enable researchers to avoid the above mentioned limitations, LifeSensors has developed the UbiQuantTM S kit as a facile, robust, and quantitative alternative to IP/WB analysis. It is built on LifeSensors UbiQuant platform, in which ubiquitinated proteins in cell lysates are captured in the wells of a precoated microtiter plate using a proprietary ubiquitin binding reagent and then detected by an antibody against the protein of interest (either native or tagged) to quantitate the amount of the protein bound. Another version of this platform – the Ubiquitin Ubiquant ELISA – opens the possibility to measure the concentration of total free ubiquitin (poly- mono-) in biological samples including cell lysates, tissue homogenates, and plasma.

Fig. 1 shows a typical experiment using the UbiquantTM S assay. Decrease in LDLR ubiquitination following treatment of transfected cells with an IDOL (E3 ubiquitin ligase) inhibitor.  Cells are treated with different concentrations of an inhibitor of the E3 ubiquitin ligase IDOL and subsequently the decreasing ubiquitination of Low density lipoprotein receptor (LDLR) is measured with the UbiquantTM S assay. More detailed results can be found in an application note recently relased by LifeSenors.

Please note that if you want to order and use this kit and intend to use a tagged-substrate, you will have to specify the epitope tag in order to receive the appropriate antibody for detection with the kit(e.g. myc, HA, FLAG®, V5, GST, etc.  Unfortunately, His6 cannot be used for this assay.) Of course you can also use your own antibodies directed against your protein of interest.

Interested to apply this method to detect the total amount of ubiquitinated proteins in your sample? Get in touch through the form below, for advice on selecting the correct anti-tag antibody you might need.

Post-translational modifications regulate Ral GTPases

RalA and RalB GTPases regulate cell motility, morphology, signaling, vesicular trafficking, and endo/exocytosis. The regulation of these functions is critical for the development and spread of cancer, implicating Ral in oncogenesis and metastasis. Both isoforms are integral for Ras-mediated tumorigenesis, metastasis, and invasion. Despite sharing 82% amino acid sequence identity, effectors, and structural/biochemical properties, RalA and RalB have their own unique functions in oncogenesis due to distinct subcellular localization and differential effector interactions. Ral localization, binding partners, and function are regulated by post-translational modifications (PTMs).

print_logoIn their recent newsletter, Cytoskeleton Inc. summarize recent findings about the relevance of geranylgeranylation, carboxymethylation, palmitoylation, phosphorylation, and ubiquitination in regulating Ral activity, subcellular localization, effector binding, and ultimately, function.

You can download a copy of this newsletter, or if you have any questions or comments, don’t hesitate to get in contact through the form below.

Kits to measure RalA activation

If you’d like to get an overview about what’s available in the small G protein field, take a look at this Small GTPase product guide.

How to manipulate and measure Autophagy?

The term Autophagy was introduced by Christian de Duve during the Ciba Foundation Symposium on Lysosomes – which was held in London in February 1963. In 1974 he was honoured with the Nobel price in Physiology or Medicine for his pioneering research about peroxisomes and lysosomes.

In this post, I’d like to give you an overview of autophagy and its implication in cell biology, and tools to manipulate and detect autophagy in cells. [Read more…]

Proteasome and (de)ubiquitination enzyme inhibition

In 2014, the U.S. Food and Drug Administration (FDA) approved the proteasome inhibiting drug Velcade (Bortezomib) for the retreatment of adult patients with Multiple Myeloma. My colleague Philippe Fixe discussed the use of Bortezomib in his post Proteasome inhibitor approved by FDA for Myeloma retreatment.

In this post, I’d like to give you an overview of the compounds which can be used to inhibit proteasome activity and furthermore the activity of De-ubiquitinases (DUB, Isopeptidases), enzymes which catalyze the de-ubiquitination of ubiquitinated proteins and which are seen as very promising drug targets especially in cancer drug development. [Read more…]

TUBEs – efficiently detect & purify poly-ubiquitinated proteins

Traditional methods to detect and purify poly-ubiquitinated proteins require either anti ubiquitin antibodies or Ubiquitin Binding Associated domains (UBAs) which display rather low affinity for Ubiquitin and show only little (if any) specificity for specific ubiquitin linkages (e.g. K63 or K48). Furthermore, these strategies require the inclusion of inhibitors of both Deubiquinating enzymes (DUBs) and Proteasome activity to protect the integrity of poly-ubiquitylated proteins, which might alter cell physiology, which in turn may negatively impact the result or introduce experimental artifacts.

To overcome these problems, LifeSensors have developed Tandem Ubiquitin Binding Entities (TUBEs). [Read more…]