Category Archives: Spotlights

Spotlights

SWATH-MS at Nature Protocols and Scientific Data

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The lab of Ruedi Aebersold recently published a Nature Protocol for generating peptide libraries for targeted analysis of SWATH MS data-independent acquisition mass spectrometry. This protocol complements a data descriptor (published in Scientific Data by the same authors) for a large-scale human assay library that can be used to support protein quantification by SWATH-MS.

In order to understand an organism’s biology and how it responds to environmental changes (e.g. disease or drug treatments), many researchers would like to be able to correctly identify and even quantify as many of the proteins in any given sample as possible. Mass spectrometry – in particular liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) – is the analytical technique most commonly used for deep and reliable exploration of the proteome.

In bottom-up proteomics, the cells of the sample are lysed, the proteins are digested into shorter peptides, the non-peptide material is removed, and the peptides separated by liquid chromatography. In LC-MS, the column separating the peptides is interfaced with a mass spectrometer and the peptides are analysed as they elute.

Sometimes the researcher will already know which proteins will be most interesting for their study; in these cases Selected Reaction Monitoring (SRM) is a very sensitive mass spectrometry approach to detecting and consistently quantifying the peptides associated with these proteins over many samples. The reactions monitored are the specific fragmentations associated with peptides-of-interest that occur in the mass spectrometer.

In many cases this information will not yet be fully available before the measurement or the number of potential protein-players in the given system might be very large. Data-independent acquisition of mass spectra for proteomics experiments is an alternative approach that extends the number of proteins that can be targeted in a sample from approximately one hundred (in SRM) to several thousand. It is made possible by the development of mass spectrometry instruments that are able to acquire high-resolutioin mass spectra at a very high sampling rate. The analysis method requires the researcher to perform initial experiments to put together a library of time and mass spectrometric coordinates corresponding to the peptides associated with each sample type. These spectra are then used as the basis for the development of highly specific assays to detect and quantify the respective peptide in subsequent samples.

We recently published a protocol for generating peptide libraries for a type of data independent mass spectrometry developed by Ruedi Aebersold and co-workers called SWATH-MS. The assay libraries are generated by collecting high-quality fragment ion spectra in data dependent acquisition mode, and processing these in a number of computational steps as shown in the workflow below.

Workflow for SWATH assay library generation

 

Scientific Data published a data descriptor for a generic large-scale human assay library to support protein quantification by SWATH-MS. This library was prepared using the procedure described in the Nature Protocol and consists of 1,164,312 transitions identifying 139,449 proteotypic peptides and 10,316 proteins; it was generated by combining the results from 331 measurements of fractions from different cell lines, tissue and affinity enriched protein samples. 

The data itself is deposited in the ProteomeXchange at the following locations:
PXD000953
PXD000954
and at SWATHAtlas

Top 100 papers of all time

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I recently gave a talk in Singapore about publishing in Nature Protocols. When I give these talks, one of the things I explain is why Nature Publishing Group is interested in publishing methodological information. For that specific talk I had great new supporting document – an article in the previous weeks Nature about the “top 100 papers”. Of course, my top 100 papers is going to be very different from yours, but the top 100 for the purposes of this article was defined as the most cited papers of all time in the Science Citation Index, owned by Thomson Reuters.

cover_nature

When I saw the cover of Nature that week, and learnt of the news story, I knew what the most cited articles were going to be – methods papers. I knew about the high cites because when we first launched Nature Protocols we looked to see which methods papers were most cited, indicative, we believed, of people having reproduced the method and being in need of a protocol. We found such papers had amazingly high cites. An example of one of the highly cited papers serving as the inspiration for a protocol was the paper by Piotr Chomczynski and Nicoletta Sacchi on isolating RNA. We felt it was important that users of the assay understood how and why it worked, and published a protocol on the assay by the original inventors.

It will be fascinating to look in ten years’ time to see the methods being invented now that have taken off and revolutionised the way we do research. I very much hope our protocols will have helped facilitate their adoption in new labs around the world.

CAUTION: Don’t mix concentrated nitric acid with organic solvents!

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Radiolabelling with copper-64 (or any other metal cation radioisotope) is done by attaching a metal chelating group to the probe of interest. In 2006, we worked with Thaddeus J Wadas & Carolyn J Anderson to publish a protocol for radiolabelling peptides with copper-64 which included a procedure for making sure that the reaction tubes and pippette tips used for the labelling were free from any other metals that might compete with copper-64 for coordination by the chelator (Box 1 in the protocol). This procedure involved washing the equipment with nitric acid, followed by rinses with ethanol followed by diethyl ether.

This is actually potentially hazardous, and the authors would like to make the following precautionary statement:

CAUTION:  When removing trace metal contaminants from pipette tips or reaction vials with 1:1 concentrated nitric acid:ddH2O, make sure no organic solvents like ethanol or diethyl ether are inadvertently mixed with the nitric acid waste.  An explosion occurred when this happened at a lab using this procedure.  Fortunately, nobody was injured.

In fact, it is possible (and advisable) to not use organic solvents for this process at all. The text for Box 1 should instead read:

BOX 1 | REMOVING TRACE METAL CONTAMINATION

Working on the tracer level requires that the reagents and vessels used be as free as possible of trace metals. To achieve this, pipette tips, reaction tubes and caps can be acid-washed by following the steps below.
Alternatively, trace metal free reaction tubes and pipette tips can be
purchased.

Removing trace metal contaminants from reaction tubes, caps and tips
1. Soak the tubes and caps or tips in a 1:1 mixture of concentrated nitric
acid and ddH2O (greater than 18 MΩ resistivity) for several hours with
periodic mixing, and then drain.
2. Rinse the tubes with ddH2O, and drain.
<CAUTION> It is recommended that ethanol and/or diethyl ether not be
used to assist in drying the tubes, as even small amounts of these
organic solvents mixed with 1:1 nitric acid ddH2O can cause an explosion.
3. Place in a container and dry in an oven at temperatures below 50 deg C.

Removing trace metal contaminants from reaction buffers and other
solutions
1. Prepare the solution(s) or buffer(s) to be used.
2. Add Chelex resin (10 g l-1) to these solutions.
3. Stir for several hours or overnight at room temperature.
4. Filter through a Corning 1-liter filter system (pore size 0.2 mm).

 

………….

Related explosions:

It will probably not surprise you to know that this is not the first time that such things have happened with nitric acid and oxidisable organics. Here are some links to related anecdotes:

Safety Chat: Nitric Acid Waste
(Lawrence Berkeley National Laboratory, 2009)

Explosion at U. Maryland: Another Nitric Acid Oopsie
(University of Maryland, 2011)

The flaming apron that sparked the invention of gun cotton and the motion picture industry
(a kitchen in Switzerland, 1845)

 

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Note added on 17 October 2013:

A corrigendum for this protocol has now been published (11 October 2013). Box 1 has been corrected in the pdf and the online version of the protocol.

https://www.nature.com/nprot/journal/v1/n6/box/nprot.2006.431_BX1.html

 

 

Model Behaviour

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Model organisms are much used in modern biology. Generally small and easy to keep in the lab, these creatures serve as the ‘typical example’ of a type of organism or biological process. Fruitflies, mice, yeast, zebra fish, Arabidopsis, etc. The list of organisms that can be regarded as models sometimes seems so long that the concept is itself undermined; how long before we see the platypus championed as a model? Nevertheless the use of ‘standard’ creatures does allow the results from different laboratories across the world to be directly compared, always assuming that these labs are treating their models in a standard way.

nematostellacoverFor the study of critical steps in animal development researchers are increasingly turning to the cnidarians, a phylum that includes jellyfish and corals, as they retain relatively simple body plans and yet are more clearly multi-cellular organisms than the somewhat colonial sponges. This week we have published four protocols concerning the cnidarian Nematostella vectensis, more commonly known as the starlet sea anemone, to act as a very basic ‘starter kit’ for anyone wishing to work with this organism.

Nematostella is a native of the east coast of the United States. It spends its time buried in mud feeding through its mouth, which is surrounded by two rings of tentacles. Slightly paradoxically they are classed as ‘vulnerable’ by the International Union for Conservation of Nature, but are also behaving as invasive aliens on the south coast of England and the west coast of the United States. But they grow fast and prolifically in the lab and have a sequenced genome1.nprot_2013_044-F5

The four protocols take us through some essential steps in working with Nematostella. John Finnerty and colleagues describe their approach to collection rearing, spawning and regeneration (doi:10.1038/nprot.2013.044)2, Isolation of DNA, RNA and proteins (doi:10.1038/nprot.2012.151)3, and characterization of RNA and protein expression (doi:10.1038/nprot.2013.014)4. To complete the quartet, Mark Martindale explains how his lab approaches overexpression and knockdown studies in this versatile beastie (doi:10.1038/nprot.2013.009)5.

We are hoping that this collection of protocols will provide a useful resource for aspiring cnidarian biologists. We would also be keen to see anyone with experience of working with Nematostella presenting their own protocols in the Protocol Exchange. The cluster also raises the following question:

Are there other emerging model organisms that are crying out for similar attention?

References:

1. Putnam NH, Srivastava M, Hellsten U, Dirks B, Chapman J et al. Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization”. Science 317, 86–94 (2007).

2. Stefanik, D. J., Lauren E Friedman, L. E. & Finnerty, J. R. Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone, Nematostella vectensis. Nature Protocols 8, 916-923 (2013).

3. Stefanik, D. J., Wolenski, F. S., Friedman, L. E., Gilmore T. D. & Finnerty,  J. R. Isolation of DNA, RNA and protein from the starlet sea anemone Nematostella vectensis. Nature Protocols 8, 892–899 (2013).

4. Wolenski, F.S., Layden, M. J., Martindale, M. Q., Gilmore, T. D. & Finnerty, J. R. Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone, Nematostella vectensis. Nature Protocols 8, 900-915 (2013).

5. Layden, M. J., Röttinger, E., Wolenski, F. S., Gilmore, T. D. & Martindale, M. Q. Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis. Nature Protocols 8, 924-934 (2013).

Preventing overfitting during the reconstruction of macromolecule images from CryoEM data

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Using cryo-electron microscopy (cryoEM) it is possible to get information about the three-dimensional structure of macromolecules. Samples can be prepared using, for example, a protocol by Grassucci et al., and EM images obtained.

What is important to note, is that the re-constructed 3D image is generated using  many thousands of  macromolecules (particles). In the figure below, for example, a total of 29,926 particles from 616 CCD frames (4k × 4k frames from a charge-coupled device camera) were used to produce the 4.3 Å resolution reconstruction shown in panel a. (taken from Zhang et al., 2010)

Image reconstruction is therefore a significant part of the process of generating meaningful data, and it is important that the averaging process doesn’t overfit the data (my understanding of this is that overfitting relates to finding patterns in the parts of the data that are really just noise; and that this over-interpretation of the data would lead to an over-estimate of the true resolution of the image).

One of our protocols for image analysis involves the use of the software XMIPP. In a recent letter to Nature Methods, Scheres and Chen introduce a script that can be used on top of the conventional projection-matching protocol in the XMIPP package to help prevent data overfitting.

The article can be accessed here:

Prevention of overfitting in cryo-EM structure determination

The script is included in the Supplementary Information (should be accessible without a site licence):

https://www.nature.com/nmeth/journal/v9/n9/extref/nmeth.2115-S1.pdf

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Nature Protocols relating to CryoEM of macromolecules:

Preparation of macromolecular complexes for cryo-electron microscopy
Robert A Grassucci, Derek J Taylor & Joachim Frank

Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes
Robert A Grassucci, Derek Taylor & Joachim Frank

SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs
Tanvir R Shaikh, Haixiao Gao, William T Baxter, Francisco J Asturias, Nicolas Boisset, Ardean Leith & Joachim Frank

Image processing for electron microscopy single-particle analysis using XMIPP
Sjors H W Scheres, Rafael Núñez-Ramírez, Carlos O S Sorzano, José María Carazo & Roberto Marabini

Cryo-EM of macromolecular assemblies at near-atomic resolution
Matthew L Baker, Junjie Zhang,Steven J Ludtke & Wah Chiu

 

 

 

 

Radiochemistry at Nature Protocols

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In March 2000 I came to England to start a PhD, and pet went from meaning a small animal you kept at home to meaning Positron Emission Tomography. There were months where my timetable was set by the half-life of iodine-124 (4.16 h), and my mind was occupied by the relative merits and problems with direct and indirect labelling of annexin V.

It has therefore pleased me to be able to commission a few protocols for radiolabeling molecules that could be used for PET or SPECT imaging.

The very first one that we published was:

Preparation of N-succinimidyl 3-[*I]iodobenzoate: an agent for the indirect radioiodination of proteins

https://www.nature.com/nprot/journal/v1/n2/full/nprot.2006.99.html

 

 

 

 

 

 

 

 

 

 

 

 

And now, in our most recent batch, we have published a group of three protocols for radiolabelling peptides and proteins with fluorine-18 using the silicon fluoride acceptor (SiFA) approach. Common to both the iodine and the fluorine radiolabelling is that a lot of effort is expended making a precursor with a labile group that will rapidly be exchanged for the radioactive ion. You want the radiolabelling reaction to go quickly and cleanly so that both the reaction and the purification can be performed in the shortest time possible maximising the amount of the radiotracer that can be used for your experiments.

The SiFA approach is almost miraculous, because it relies on the fact that for this extraordinary moiety fluorine-18 will replace the stable isotope, fluorine-19. Shown below is the reaction scheme for labelling peptides.

 

The three SiFA protocols are:

One-step 18F-labeling of peptides for positron emission tomography imaging using the SiFA methodology
https://www.nature.com/nprot/journal/v7/n11/full/nprot.2012.109.html

Synthesis of [18F]SiFB: a prosthetic group for direct protein radiolabeling for application in positron emission tomography
https://www.nature.com/nprot/journal/v7/n11/full/nprot.2012.110.html

Protein labeling with the labeling precursor [18F]SiFA-SH for positron emission tomography
https://www.nature.com/nprot/journal/v7/n11/full/nprot.2012.111.html

 

Here is a complete list of Nature Protocols that involve radiolabelling with isotopes suitable for PET or SPECT imaging:

Carbon-11, PET

Labeling of aliphatic carboxylic acids using [11C]carbon monoxide
https://www.nature.com/nprot/journal/v1/n2/full/nprot.2006.112.html

Tagging recombinant proteins with a Sel-tag for purification, labeling with electrophilic compounds or radiolabeling with 11C
https://www.nature.com/nprot/journal/v1/n2/full/nprot.2006.87.html

 

Fluorine-18, PET

One-step 18F-labeling of peptides for positron emission tomography imaging using the SiFA methodology
https://www.nature.com/nprot/journal/v7/n11/full/nprot.2012.109.html

Synthesis of [18F]SiFB: a prosthetic group for direct protein radiolabeling for application in positron emission tomography
https://www.nature.com/nprot/journal/v7/n11/full/nprot.2012.110.html

Protein labeling with the labeling precursor [18F]SiFA-SH for positron emission tomography
https://www.nature.com/nprot/journal/v7/n11/full/nprot.2012.111.html

Synthesis of N-succinimidyl 4-[18F]fluorobenzoate, an agent for labeling proteins and peptides with 18F
https://www.nature.com/nprot/journal/v1/n4/full/nprot.2006.264.html

Preparation of 18F-labeled peptides using the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition
https://www.nature.com/nprot/journal/v6/n11/full/nprot.2011.390.html

PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG
https://www.nature.com/nprot/journal/v1/n6/full/nprot.2006.459.html

Molecular PET imaging of HSV1-tk reporter gene expression using [18F]FEAU
https://www.nature.com/nprot/journal/v2/n2/full/nprot.2007.49.html

 

Copper-64, PET

Preparation of carbon nanotube bioconjugates for biomedical applications
https://www.nature.com/nprot/journal/v4/n9/full/nprot.2009.146.html

Radiolabeling of TETA- and CB-TE2A-conjugated peptides with copper-64
https://www.nature.com/nprot/journal/v1/n6/full/nprot.2006.431.html

 

Gallium-68, PET

Facile radiolabeling of monoclonal antibodies and other proteins with zirconium-89 or gallium-68 for PET imaging using p-isothiocyanatobenzyl-desferrioxamine
https://www.nature.com/protocolexchange/protocols/412

Conjugation of DOTA-like chelating agents to peptides and radiolabeling with trivalent metallic isotopes
https://www.nature.com/nprot/journal/v1/n2/full/nprot.2006.175.html

 

Yttrium-86, PET

Conjugation of DOTA-like chelating agents to peptides and radiolabeling with trivalent metallic isotopes
https://www.nature.com/nprot/journal/v1/n2/full/nprot.2006.175.html

 

Zirconium-89, PET

Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine
https://www.nature.com/nprot/journal/v5/n4/full/nprot.2010.13.html

 

Technetium-99m, SPECT

Radiolabeling of HYNIC–annexin V with technetium-99m for in vivo imaging of apoptosis
https://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.17.html

Methods for MAG3 conjugation and 99mTc radiolabeling of biomolecules
https://www.nature.com/nprot/journal/v1/n3/full/nprot.2006.262.html

An improved synthesis of NHS-MAG3 for conjugation and radiolabeling of biomolecules with 99mTc at room temperature
https://www.nature.com/nprot/journal/v2/n4/full/nprot.2007.144.html

 

Indium-111, SPECT

Conjugation of DOTA-like chelating agents to peptides and radiolabeling with trivalent metallic isotopes
https://www.nature.com/nprot/journal/v1/n2/full/nprot.2006.175.html

 

Iodone-124 / Iodine-125, PET / SPECT

Preparation of N-succinimidyl 3-[*I]iodobenzoate: an agent for the indirect radioiodination of proteins
https://www.nature.com/nprot/journal/v1/n2/full/nprot.2006.99.html

Label-free proteomics – the protease matters

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Trypsin is the most commonly used protease for mass spectrometry–based proteomics experiments, because of its well defined specificity (it results in peptides with either lysine or arginine at the C-termini).

With an increasing focus on answering the question “How many copies of this protein are present per cell?”, it becomes important to make sure that the protease digestion does not result in more- or fewer-than-expected peptides for any given protein.

Is trypsin always the best protease for such proteomics experiments?

It turns out that it would be a good idea to try other proteases as well.

In a letter to Nature Methods, Peng et al. describe results from an experiment where they treated aliquots from a yeast lysate with different proteases (trypsin, Lys-C,  Lys-N and chymotrypsin). They then performed strong cation exchange chromatography followed by LC–MS/MS and generated proteomics data sets based on both spectral counts and ion volume. While there was a good correlation between the results obtained from technical replicates, correlation between samples obtained using different proteases was less good, an effect which was especially pronounced with chymotrypsin, which has a different cleavage specificity.

The authors found that while amount of some proteins was similar no matter what protease was used (e.g. those shown in (i) below), there were others where using trypsin seemingly over-estimated the copy number (ii) OR underestimated the copy number (iii), and there were proteins which would not have been seen if only one of the proteases had been used (iv).

(the Supplementary information provides a lot of information regarding the methods used, and includes a more comprehensive set of results).

 

 

Mao Peng, Nadia Taouatas, Salvatore Cappadona, Bas van Breukelen, Shabaz Mohammed, Arjen Scholten & Albert J R Heck. Protease bias in absolute protein quantitation. Nature Methods 9, 524-525 (2012)

 

Protocols relating to the Structure and Composition of Plant Cell Walls

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People have already exploited the physical properties of plant cell walls in, for example, the production of paper, textiles and building materials. In the future it may be possible to engineer plants to produce other environmentally friendly / biocompatible materials with different performance characteristics.

In addition, plant cell walls are potentially a very large source of carbohydrates for biofuels, and methods to better understand the ultrastructural and chemical barriers to their digestibility, as well as to monitor responses to different experimental treatments, would be very valuable in this research.

Over the past month, we have published five protocols that relate to plant cell walls: their structure and composition, and the biochemistry underlying their development. I have created a webpage where these, and any future protocols that we might publish on this subject, can be collated.

Protocols relating to Plant Cell Walls

In a sense this series on plant cell walls is a return home. The department in which I did the Cell Biology part of my undergraduate degree was very much focussed on plant biology, and TEM of recalcitrant seeds was part of the course. There was, therefore, a time when the walls of my room were littered with little printouts showing images of cells walls and sections of endoplasmic reticulum that had patterns that somehow pleased me. I had a hand-wavey “understanding” of how cell walls formed, and some sort of feeling for the fact that this was not a trivial process.

Admittedly all of this was rather a long time ago, and I suppose is only interesting if you happen to think of me solely as a chemistry-type!

It does, however, give me the small amount of credibility to enable me to say that almost everything that I learnt at University in this subject area is now out of date. In fact, it is almost completely pointless for me to mentally refer back to my undergraduate biology courses as a source of information or inspiration. This is not really because the information that that I was taught was wrong, but that the mental conclusions I made were based on an incomplete picture. This can be exemplified by the fact that there were two things about the title of the following article by Nottburger Gierlinger and Manfred Schwanninger –

Chemical Imaging of Poplar Wood Cell Walls by Confocal Raman Microscopy

– that somehow surprised me:

– There is something about the chemistry of plant cell walls that people are finding very interesting
– Raman spectroscopy has made the transition to being a microscopy technique

If people are looking at plant cell walls using confocal Raman microscopy (which is surely on the super-cool end of the techniques spectrum), they must be looking at it using other more familiar techniques as well.

Further investigation convinced me it would be a good idea to commission a collection of protocols covering a variety of approaches that can be taken to gain a better understanding of plant cells walls. I got in touch in Professor Geoffrey Fincher, and I am very grateful for his help in shepherding the preparation of four additional protocols. I would also very much like to thank the authors for all their hard work and patience; I am very pleased with these protocols and hope that they will be interesting and useful to other researchers.

These are the five protocols that resulted from this process:

Whole Plant Cell Wall Characterization using Solution-state 2D-NMR
Shawn D. Mansfield, Hoon Kim, Fachuang Lu, and John Ralph
https://www.nature.com/nprot/journal/v7/n9/full/nprot.2012.064.html

Determining the polysaccharide composition of plant cell walls
Filomena A Pettolino, Cherie Walsh, Geoffrey B Fincher & Antony Bacic
https://www.nature.com/nprot/journal/v7/n9/full/nprot.2012.081.html

Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis
Christian Brown, Felicia Leijon & Vincent Bulone
https://www.nature.com/nprot/journal/v7/n9/full/nprot.2012.089.html

Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes
Sarah M Wilson & Antony Bacic
https://www.nature.com/nprot/journal/v7/n9/full/nprot.2012.096.html

Imaging of plant cell walls by confocal Raman microscopy
Notburga Gierlinger, Tobias Keplinger & Michael Harrington
https://www.nature.com/nprot/journal/v7/n9/full/nprot.2012.092.html

Top Exchange Protocols – By number of views

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The three most viewed Exchange Protocols appear on the Protocol Exchange homepage; they don’t seem to change much, mostly because they are always highlighted!

This blogpost will list the top 13 Exchange Protocols (it is after all the 13th of July!) to give a better picture of which protocols people find most interesting, and to show our appreciation for some the researchers who upload excellent protocols to our site.

Of course, that they are the most downloaded does not necessarily mean that they are the ones that will be most useful to you. To find protocols relevant to your research, try using the browse function on our site.

The list below was obtained using Webtrends, and reflects the order of protocols in terms of the number of views over the last 91 days.

 

1.  A protocol for in vivo detection of reactive oxygen species

2. Neural Stem Cell Culture: Neurosphere generation, microscopical analysis and cryopreservation

3.  Production of neuron-preferential lentiviral vectors

 

4.  Western blot analysis of sub-cellular fractionated samples using the Odyssey Infrared Imaging System

Yukiko Misawa, Ying Li, David Rekosh & Marie-Louise Hammarskjold

5.  Fluorescence in situ hybridization (FISH) for DNA replication origins

Patrizia Gasparini & Omar Malazzi

6.  COBRA Toolbox 2.0

Daniel Hyduke et al. from Bernhard Palsson’s lab

7.  Trans-well migration assay

Ofer Mandelboim

8. Measuring fatty acid oxidation in tissue homogenates

Matthew D Hirschey & Eric Verdin

9.  Chromatin immunoprecipitation (ChIP) assay

Zhongfu Ni, Danny W.-K. Ng, Jianxin Liu & Z.Jeffrey Chen

10.  Combination of chemical cross-linking and pull-down assay to study transient protein-protein interactions

Feng Gong, Deirdre Fahy & Michael J. Smerdon

11.  Immunohistochemistry and in situ hybridization protocols

Joshua Hunsberger & Samuel Newton

12.  Simultaneous detection of murine antigen-specific intracellular cytokines and CD107a/CD107b by flow cytometry

Joyce J. Ruitenberg, Smita A. Ghanekar, Dirk G. Brockstedt  & Holden T. Maecker

13. A simple and rapid method for RNA isolation from plant tissues with high phenolic compounds and polysaccharides

Kam-Lock Chan, Chai-Ling Ho, Parameswari Namasivayam & Suhaimi Napis

 

 

From lipids to membranes

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Membranes are amazing things: they surround the cell, the nucleus, and many other intracellular organelles. They keep stuff inside, they keep other stuff out. There are protocols for extracting them from cells, and there are protocols for making them from scratch.

Preparing them “artificially” has the obvious advantage that you can exquisitely control their composition and properties, and a number of assays looking at membrane function and behaviour start with this step.

Short aside

For some reason these protocols always remind me of a certain German physicist who made some scientific observations that started at the kitchen sink. I can never remember her name or any of the details, but the miracle of the internet allowed me pin down her name: ‘Agnes Pockels’, and that the experiments related to detergents and surface tension. If you have access to Nature, then you can read some of the letters that she published in the 1890s:  “Surface Tension” (The text of this letter can be accessed here as well. Lord Rayleigh asked Nature to publish a letter that she had written to him and for some reason this makes me feel quite emotional. ), “Relations between the surface-tension and relative contamination of water surfaces” and “On the spreading of oil upon water“.

Back to the present, and back to the topic!

Here is a taste of some of the experimental possibilities covered in Nature Protocols so far.

Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells
Erdem Karatekin & James E Rothman

In this protocol, you make both a planar supported bilayer (SBL) and some small unilamellar vesicles (SUV).

 

The SBL is put together in a microfluidic flow channel…

…and docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorescence microscopy.

 

A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins
Jiajie Diao, Yuji Ishitsuka, Hanki Lee, Chirlmin Joo, Zengliu Su, Salman Syed, Yeon-Kyun Shin, Tae-Young Yoon & Taekjip Ha

Here, the assays are looking at the fusion of vesicles in solution with vesicles velcro’ed (biotin-neutravidin) to the bottom of a flow chamber. Both assays involve FRET pairs: either paired-dyes in reacting membranes or on the two ends of a DNA hairpin.

 

Generation of phospholipid vesicle-nanotube networks and transport of molecules therein
Aldo Jesorka, Natalia Stepanyants, Haijiang Zhang, Bahanur Ortmen, Bodil Hakonen & Owe Orwar

This protocol starts by taking a soybean polar extract and using it to prepare giant unilamellar vesicles (GUV) attached to multilamellar membrane reservoirs. By some very fancy manipulations, it is possible to make networks of vesicles connected by nanotubes that can be used for experiments relating to transport of molecules.

 

An earlier post on Membrane Proteins is related summary; and other protocols can be found by browsing our content.