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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8 criteria for selecting your ELISA kits

Biomarkers specialists are often asked to select an ELISA kit for researchers: with thousands of ELISA references available on the market, the choice can be tricky regarding proteins for which several kits available.

When researchers have to choose a new ELISA kit, the price is regularly the first parameter of selection. But my experience with long term projects shows that it should in fact be the very last one…

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Discovery of new biomarkers… 3 tips regarding controls

One of the recurrent questions that we get at the Biomarkers team at tebu-bio is on what controls should be included in a given experiment. Either if the experiment is done by researchers in their lab, or if we collect their samples and perform the analysis in our lab, a good design starts by using the most convenient controls.

One of the controls is related to the study itself. In this sense, definition of what a control population is, and how we want to study it vs. a cohort of patients has been discussed elsewhere in a proteomics post. Today, we will put our spotlight on the “technical” controls, i.e. those related to the technique itself.

Control # 1 – positive control

Obvious. We need to check that the technology we are using is able to detect what we want to detect. And before starting with unknown samples, we need to check that it works in samples we know well.Multicoloured wells - Blog Thumbnail

Ideally, a positive control should be as similar as the samples we want to analyse. In this sense, samples for a given health state (be it with a disease or not), are commercially available, or they can be found if not yet available. tebu-bio has a network of collaborations with private companies that can provide validated samples, fulfilling all ethical and clinical criteria.

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Background can be an issue…only if you are not able to detect it. If you can see it, then you can either modify your protocol, or discard that sample. Picture shows an example of what can be seen with slightly-hemolysed plasma.

Alternatively, we can use recombinant or chemically synthesised controls. If we take the example of recombinant proteins (either in pure form or spiked in biological samples), there are many which are commercially available, or they can be made upon demand. Here it is important that the recombinant protein is very similar to the one found in an organism, including glycosylations and other post-translational changes. In this sense, for many control proteins, HEK293 is preferred over E.coli as an expression system.

Control #2 – negative control

Water. Or PBS. Or not?

Ideally, a negative sample should be as similar as to our case samples as possible. Meaning that it has the same clinical and biological parameters than our samples of interest…

Commercially available samples mentioned previously can be a good approach. For immunological studies involving cell culture supernatants, it is important to include a control with the culture medium only, as FBS can affect the specificity of the assay and can render false-negatives due to background.

Background due to FBS is not detected by technologies such as ELISAs or bead-based, whereas it is detected in optical-based technologies such as arrays and Q-plex.

Control #3 – technical replicates

Every technology has an inherent coefficient of variation (CV).

Genomic technologies usually are under 5 % CV. Immunoassays are around 10 to 25 % (or even more). This means that, for some biomarkers where the difference between healthy vs. disease is small, CV may hide the relevance of these biomarkers. This is especially dramatic in studies related to signal transduction, where differences are usually very small.

6-plicates in an antibody array.

6-plicates in an antibody array.

A way to make sure about whether a result comes from real biology or artificial CV is the performance of replicates.

Triplicates (or even 4-plicates) have been popular with ELISA users. Nowadays, however, most researchers perform duplicates, and repeat the analysis of the sample if the results are very discordant. This approach is quite practical, and still allows to have accurate results in a sensible way (i.e. not doing 4-plicates for every sample!).

Antibody arrays in the market usually have replicates spotted onto the same slide (from duplicates to 8-plicates), which can be considered as semi-independent technical replicates. Therefore, there is no need, in most cases, to perform additional technical replicates.

In any case, every project is different, so we are continously advising our customers on what is the best approach for one given study. From its design to the technology best suited to get the best results, we are glad to contribute to the advance of the understanding of biomarkers in several diseases.

Wondering what controls to include in your experiment? Don’t hesitate to contact us!

More is not always better – Tech tips for ELISAs

Following our previous post on how to improve results obtained in ELISA, let’s focus today on one specific point, which is reducing background.

ELISA has many advantages, but one of the drawbacks is that, since we cannot “see” how the reaction works (in contrast to other optical-based technologies such as antibody arrays or Q-plex), high final Abs values may come from a specific signal… or be due to background.

Usually, incorporating sufficient controls in the ELISA plate will allow users to discriminate real positives from false positives (e.g. if you are using cell culture supernatant with FBS % over 1 %, it might be wise to include a medium-only control). FBS contains cytokines that can cross-react with antibodies, even if targeted to different species, in about 10 % of the cases (based on our experience at the Biomarkers team at tebu-bio).

Anyway, if you suspect that you are obtaining a high background in your ELISA, and would like to improve it for future experiments, be sure to follow these guidelines (thanks to Daniel at Raybiotech, Inc. for helpful tips & tricks!). [Read more…]

Flow cytometry intracellular staining

Intracellular Flow Cytometry enables the identification and analysis of signaling and functional markers within cells from samples containing heterogeneous cell populations. Many cell types can be thus identified through their intracellular Flow Cytometry patterns and key intracellular signaling pathways and their respective post-translational modifications can be conveniently analysed. Behind these unique performances, Intracellular Flow Cytometry require anyhow optimized immunoreagents for cell permeabilization, intracellular staining and fluorescent detection. [Read more…]

5 tips for working with unstable ORF expression clones

Mammalian expression constructs for certain genes such as human c-KIT are notorious for undergoing frequent recombinations during cloning and transformation steps in molecular biology labs. Experts suggest that certain genes are “toxic” to bacteria thus leading to a situation in which recombined plasmids are favored. While the molecular mechanisms for this toxicity may be unclear, the end result is that efforts to amplify, subclone, mutate, or make derivative vectors often result in a new plasmid with unwanted sequence errors.

Online science discussion forums such as ResearchGate include a variety of strategies proposed by researchers experiencing this kind of plasmid instability. Suggestions include advice such as culturing the bacteria at room temperature rather than 37°C, culturing on plates rather than in flasks, using low copy number EPI400 competent cells, picking the small colonies rather then the large ones from the LB plate, replacing the ampicillin resistance cassette to prevent satellite colonies (so you can see the small colonies), or using a Gateway vector.

But which methods work best?

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5 Tips for Flow Cytometry

Flow cytometry, even if somehow a “classical” technique, is still very valuable in Immunology. Uses of flow cytometry vary from cell identification & sorting based on membrane molecules, characterisation and quantification of secreted and intracellular molecules, as well as, more recently, analysis related to exosomes.

Following our series of technical tips for common experiments in Life Sciences (phospho-WB, secondaries, IF, ELISA, primary cell culture), we present today a series of tips to improve your flow cytometry results. [Read more…]

The gold rush for better diagnostics

Back in the XIXth century, tens of thousands of people rushed to California to find gold and become rich. Most of them did not succeed, but those who did, really did (especially the companies owning the mines). Something similar happens with diagnostic kits nowadays. The trend is towards more personalised medicine, either at the diagnosis step, or at the therapeutical step. Newly marketed drugs include companion diagnostics, development of new diagnostics is increasing, and the need for accurate results is more important than ever. One size does not fit all.

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PrimerGenesis an online tool for today’s molecular biologists

Now that custom gene synthesis costs have made ordering a DNA plasmid with any desired sequence extremely affordable, it is difficult to see the importance of teaching young molecular biologists how to design primers for site directed mutagenesis, adding tags, or C-terminal truncations. I recall during my PhD how our lab’s molecular biology guru tried to teach me how to design mutagenesis oligos to introduce a novel restriction site and how important the GC-clamp was.

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Useful tip for better zebrafish whole-mount IHC staining

The zebrafish (Danio rerio) is a common vertebrate model organism in scientific research for studying vertebrate development, gene function, and regenerative abilities. Interestingly, this convenient model is compatible with large-scale genetic analysis for developmental & regenerative biology, oncology, environment & toxicology, neurobiology…

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