Very Frequently Asked Questions

General & practical info | MASS SPECTROMETRY based workflows | Capillary Western (JESS)
 

General & practical info

How to proceed to set up a study or experiment ?

Contact us by email or phone to discuss your project or experiment. For more in depth discussions we can organize a meeting in person or via zoom. Unless you have previous experience of proteomics experiments, it is crucial that we discuss experimental design and sample preparation BEFORE you proceed with your experiments. This will help minimize failure and frustration. Very often, we help you to plan a pilot experiment that is usually crucial to assess feasibility.

PI's preparing a grant application should contact us well in advance of the deadline to discuss the project and establish the necessary budget.

What techniques do you offer ?

Mass spectrometry is our main technology that we apply for untargeted protein and proteome analyses. We also offer targeted protein detection by capillary western assays.

Where can I find more technical information or tutorials ?

Visit our Downloads page.

I have samples ready, how do I submit them for analysis ?

Fill in the on-line form (https://paf-sample-submission.unil.ch), and tell us by e-mail when you want to bring the samples.

MASS SPECTROMETRY based workflows

Which type of samples can be analysed ?

We can process almost any type of sample: cultured cells, biological tissues, protein solutions. In many cases we can do the extractions ourselves, unless you have specific experience. We have worked with many types of organisms and sorts of samples. However, we cannot guarantee that we will have a good recipe for everything !

How much protein material is needed ?

This depends critically on the biological question and the type of sample to analyse. For "normal" total proteome profiling (e.g. human cell lines), 2 x 10e5 cells are typically sufficient, equivalent to approximately 20 ug of total protein. Cell numbers may vary a lot depending on the size of cells. Extraction of tissues requires at least 1-2 milligrams wet tissue (mammalian tissues) or 5-10 mg for plant tissues. For other types of material one should plan to have at least 50 ug net of protein material.  Of course, having some spare sample is always recommended.

How many proteins can be profiled ?

This depends on the complexity and dynamic range of the sample (= distribution of abundances ). Up to 8000 proteins can be profiled in standard cultured cell lines in ideal conditions. In other samples, dominance by a few molecular species (ex. actin/myosin in muscle tissue or RUBISCO in plants) can strongly limit the proteome coverage obtainable within reasonable measurement times. Nevertheless, meaningful data can be produced in these samples, too. Numbers of proteins identified in interactomics experiments can vary a lot, between a few hundreds to 3000-4000 depending on sample complexity and stringency of purification.

Do you analyse PTM's ?

We support large scale analyses of phosphoproteome and ubiquitinome. Other PTMs can often be  analysed on defined target proteins, if appropriate mass spectrometry methods exist.

What is the maximum number of samples in a project ?

In routine work we will consider projects including up to 30 samples. Larger projects are not excluded but require specific planning and discussion.

How long will it take to complete the analyses ?

"Small" experiments with less than 6-8 samples and direct processing can be carried out in 10-14 days. However this strongly depends on the workload that we are experiencing. This tends to vary strongly during the year, often in unpredictable manner.

What type of results will we receive ?

At the minimum you will receive a table with a list of proteins (or modification sites in case of PTM's) and columns with associated quantitative values. Some overall statistics across conditions and a basic pathway enrichment analysis will be included for quantitative experiments with replicates.

Is the facility taking care of downstream bioinformatics analysis ?

We can do some more advanced data processing and analysis. But we are not specialists in bioinformatics and we will only take you to a certain point. For multivariate statistics, clustering or in general more sophisticated data analyses we will redirect you to other core facilities at UNIL. Ultimately, the interpretation of your data is up to you.

Capillary Western (JESS)

What is this technology for ?

JESS is made to detect and quantify proteins and or protein/isoforms in biological samples whenever a specific antibody is available. The technique is similar to a western blot, with everything performed into capillaries.

What are its advantages relative to classical Western Blot ?

JESS run requires lower amounts of proteins and antibodies than a classical western blot with everything performed in less than 4 hours. Furthermore, JESS is fully automated and thus less prone to technical bias as compared to western blot. JESS performs well with low and high molecular weight proteins since there is no transfer (proteins are bound to the capillary walls after migration). Capillaries being independent, 24 or 12 combinations of samples and antibodies can easily be done in parallel. Total protein normalization or a second immunodetection (for precisous samples) can also be performed in the same run.

What are the sample requirements for JESS ?

Most of the classical protein extractions are compatible with a JESS run (RIPA, Laemmli buffer). Depending on the protein targets, different sample concentrations are required. Classicaly, 1 ug of protein and 0.2 ul of primary antibody are required per capillary.

How are JESS runs organized ?

A JESS run can be done with 12 or 24 independent samples. Just bring your samples and/or the primary antibodies. We do provide antibodies against some common targets.

For quantitative detection, a preliminary test run has to be performed to detect the linearity range of the sample and antibody response, as well as to validate the combination of different antibodies whenever required.

Which proteins could be detected by JESS ?

Each time there is an antibody directed against one particular protein/protein isoform, they can be detected by JESS. Depending on the protein molecular weight, one of the 3 matrices could be used: 2-44 kDa, 12-230 kDa, 66-480 kDa.

GĂ©nopode - CH-1015 Lausanne
Switzerland
Tel. +41 21 692 56 76
Fax +41 21 692 57 05