Bronwen is off to Cambridge for a Synthesis in Organic Chemistry symposium

Today, I am in a good mood, because I know that next week I am going to a conference! As much as I love looking at manuscripts and browsing through the literature for commissioning ideas, it will be good to be out of the office and listening to scientists talking about their work. Hopefully, I will get to meet and chat to a few as well! I will be staying in a little bed-and-breakfast in the evenings, and during the day I will attend talks by eminent chemists at the 22nd International Symposium: Synthesis in Organic Chemistry in Cambridge.

Is anyone else going to the meeting? If so leave a comment and I’ll make sure to look out for you. Or just come and say hello when you are there.

To highlight our chemistry content, the following protocols have been made open access until the end of July.

Protocols relating to metathesis:

Synthesis of N-heterocyclic carbene ligands and derived ruthenium olefin metathesis catalysts

Xavier Bantreil & Steven P Nolan

Synthesis of all-hydrocarbon stapled α-helical peptides by ring-closing olefin metathesis

Young-Woo Kim, Tom N Grossmann & Gregory L Verdine

Solid-phase synthesis of short α-helices stabilized by the hydrogen bond surrogate approach

Anupam Patgiri, Monica Z Menzenski, Andrew B Mahon & Paramjit S Arora

Olefin cross-metathesis-based approaches to furans: procedures for the preparation of di- and trisubstituted variants

Timothy J Donohoe, John F Bower & José A Basutto

Click chemistry

Alkyne-azide click reaction catalyzed by metallic copper under ultrasound

Pedro Cintas, Alessandro Barge, Silvia Tagliapietra, Luisa Boffa & Giancarlo Cravotto

More Click Chemistry protocols can be found here

Enzyme catalysed

Preparative scale Baeyer-Villiger biooxidation at high concentration using recombinant Escherichia coli and in situ substrate feeding and product removal process

Iris Hilker, Maria C Gutiérrez, Roland Furstoss, John Ward, Roland Wohlgemuth & Véronique Alphand

Other useful reactions

Pd-catalyzed Suzuki-Miyaura reactions of aryl halides using bulky biarylmonophosphine ligands

Ryan A Altman & Stephen L Buchwald

The partial reduction of electron-deficient pyrroles: procedures describing both Birch (Li/NH3) and ammonia-free (Li/DBB) conditions

Timothy J Donohoe & Rhian E Thomas

The use of the Ugi four-component condensation

Stefano Marcaccini & Tomás Torroba

Synthesis of specific reagents

A practical guide to the synthesis of dinitroindolinyl-caged neurotransmitters

Graham C R Ellis-Davies

A concise and scalable route to L-azidohomoalanine

Stefanie Roth, William C Drewe & Neil R Thomas

Analytical techniques

A method for concurrent diazomethane synthesis and substrate methylation in a 96-sample format

Lana S Barkawi & Jerry D Cohen

Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates

Sherif S Ebada, Ru Angelie Edrada, Wenhan Lin & Peter Proksch

Mosher ester analysis for the determination of absolute configuration of stereogenic (chiral) carbinol carbons Thomas R Hoye, Christopher S Jeffrey & Feng Shao

A Finer Point relating to Nature Protocols RSS feeds

Following on from the previous post, I would like to show you what else you can do to customise your Nature Protocols RSS feeds.

If you have a look at the list of RSS feeds for the categories, you will notice that all of the links have “.rss” between “protocols” and the question mark.

For example:

Spectroscopy

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Spectroscopy

Structural Biology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Structural+biology

Synthetic Chemistry

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Synthetic+chemistry

What is really cool, is that you can make an rss feed from any search term (or group of search terms) accessible from the Browse page.

For example, if you are very interested in NMR, you might search for this term within the Structural biology category, insert “.rss” into the webaddress and then copy the resulting link into your feedreader!

Webaddress for the search results:

http://www.nature.com/protocolexchange/protocols?commit=Go&protocol_search%5Bfacets%5D%5Bcategory%5D=Structural+biology&protocol_search%5Bq%5D=NMR

Webaddress for the RSS feed:

http://www.nature.com/protocolexchange/protocols.rss?commit=Go&protocol_search%5Bfacets%5D%5Bcategory%5D=Structural+biology&protocol_search%5Bq%5D=NMR

Another example application:

If you are collaborating with other research teams and want to be able to easily group protocols that “belong” to the collaboration, you could agree on a unique connecting word or phrase to add to the keywords when uploading your Exchange Protocols. You would be able to use this term to easily find the protocols, plus you could have an RSS feed to let you know when more protocols had been added.

RSS Feeds at the Category Level – Hoorah!!

Ever since I found out about RSS feeds (and igoogle), I have looked at our home page and thought: “Wouldn’t it be nice if there were RSS feeds at the category level?”.

So if this is the one thing that has been missing from your life all these years, I have some good news: You can do it now!

Unfortunately, we don’t have the little orange buttons yet, but if you copy one of the links below into your feedreader it should work.

Biochemistry

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Biochemistry

Cell biology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Cell+biology

Cell culture

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Cell+culture

Chemical modification

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Chemical+modification

Computational biology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Computational+biology

Developmental biology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Developmental+biology

Epigenomics

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Epigenomics

Genetic analysis

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Genetic+analysis

Genetic modification

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Genetic+modification

Genomics

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Genomics

Imaging

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Imaging

Immunological techniques

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Immunological+techniques

Isolation, Purification and Separation

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Isolation%2C+Purification+and+Separation

Metabolomics

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Metabolomics

Microbiology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Microbiology

Model organisms

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Model+organisms

Nanotechnology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Nanotechnology

Neuroscience

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Neuroscience

Nucleic acid based molecular biology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Nucleic+acid+based+molecular+biology

Pharmacology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Pharmacology

Plant Biology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Plant+biology

Protein analysis

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Protein+analysis

Proteomics

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Proteomics

Spectroscopy

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Spectroscopy

Structural Biology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Structural+biology

Synthetic Chemistry

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Synthetic+chemistry

Tissue Culture

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Tissue+culture

Toxicology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Toxicology

Virology

http://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bcategory%5D=Virology

Membrane protein protocols

While browsing recently published papers in other journals, I came across Supramolecular fishing for plasma membrane proteins using an ultrastable synthetic host-guest binding pair (published in Nature Chemistry). The idea here is that proteins on the surface of the cell are chemically modified by reaction with 1-trimethylammoniomethylferrocene (AFc). After cell lysis, the modified proteins bind to beads coated with cucurbit-7-uril. Cucurbit-7-uril and AFc interact to form an ultrastable host-guest binding pair. The captured proteins are then recovered either by treatment with a strong competitor or by heating to 95 C in buffer conditions.

I thought that this was rather pleasing! It reminded me of a protocol we had published on the metabolic labeling of glycans with azido sugars, and got me to thinking: “What sorts of protocols do we have for looking at membrane proteins?”.

Using our Browse functionality plus a PubMed search I came up with 17 that I thought would be relevant (please forgive me (and let me know!!) if I have missed something vitally important). These protocols can be roughly divided into those working with a specific protein that you already know (the majority), and those where you are working with membranes from whole cells. When looking at the structure and function of purified membrane proteins, one of the main challenges seems to be the design of a matrix such that you can get meaningful data from proteins that are in conditions as similar as possible to the cell membrane itself.

These are some notes that I made while going through the protocols:

Expression and purification

Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems

- This protocol includes methods for preparing E.coli s30 extracts and T7RNAP.

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

- This protocol includes a method for transforming a gene-vector construct into S. cerevisiae.

- Purification is via a His-tag

Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase

Looking mostly at structure

A general protocol for the crystallization of membrane proteins for X-ray structural investigation

- His-tagged proteins are expressed in E.coli. The membranes are isolated and solubilised. Purification on a nickel column is followed by removal of the his-tag and size exclusion chromatography. Crystallisation is by hanging-drop vapour diffusion.

Crystallizing membrane proteins using lipidic mesophases

Bicelle samples for solid-state NMR of membrane proteins

- Isotope labelled proteins are analysed in bicelles. The lipid bilayers of these are aligned perpendicular to the magnetic field forming a liquid crystalline phase.

Synthesis of TOAC spin-labeled proteins and reconstitution in lipid membranes

- The protein is synthesised via FMOC chemistry. When analysed by electron paramagnetic resonance, the TOAC residue accurately reports the position, orientation and dynamics of the peptide backbone near the labelled site.

Atomic force microscopy and spectroscopy of native membrane proteins

- The authors use purple membrane from Halobacterium salinarum as an example. The stylus of the AFM is used to obtain the topography of the membrane as well as to mechanically manipulate the protein. Measurements taken in the process of unfolding the protein provide information regarding the molecular interactions within it.

The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology

- This protocol uses GFP-fusion proteins. The link will take you to a cartoon showing a single cell before and after exposure to digitonin and trypsin.

Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins

- Each residue in the protein is systematically mutated to cysteine. The proteins are reacted with [N-ethyl-1-^14^C]ethyl-maleimide, separated by SDS-PAGE and analysed using a PhosphoImager.

Looking mostly at function using specific proteins

Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+-ATPase as an example

- This method uses the radioactive detergent ^14^C-n-dodecyl-β-D-maltoside. The Stokes radius is determined either by size-exclusion chromatography or by ultracentrifugation sedimentation velocity analysis.

Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling

Expression cloning and radiotracer uptakes in Xenopus laevis oocytes

- This protocol is at the border of a few sections as it uses a functional assay (uptake of a radiotracer appropriate to the type of membrane protein that you are interested in) to either find new proteins or work what mutations will result in changes in transport activity.

- cRNA is injected into the oocytes.

Two protocols that I wasn’t certain how to classify

The preparation and development of cellular membrane affinity chromatography columns

- The membrane proteins are isolated by centrifugation (usually from a transfected cell line containing a target protein) and attached to a stationary phase.

- Frontal affinity chromatography using labelled ligands is used to measure molecular interactions.

Detecting interactions with membrane proteins using a membrane two-hybrid assay in yeast

- This protocol is for the MYTH system shown in the figure below.

Proteomic-type protocols

Quantitative proteomic approach to study subcellular localization of membrane proteins

- The method used is called LOPIT (localisation of organelle proteins by isotope tagging).

- Cell membranes are separated into fractions by equilibrium density gradient centrifugation. Each fraction is labelled using a different iTRAQ reagent, and the samples are pooled.

Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS

- The key features of this protocol are (1) the use of 60 % methanol to dissolve the membrane-protein sample, and (2) that the strong cation exchange chromatography is done off-line to allow for optimisation of the fractionation to improve the capacity of the LC-MS/MS analysis. This is significant, because it allows the identification of 1500-2500 unique proteins.

An introduction to Lab Groups

A feature of the new, improved Protocol Exchange site is that each protocol is associated with a Lab Group. A full list of these Lab Groups can be access from the Lab Groups tab.

outstanding labgroup 3.PNG

Lab groups can be:

1. Closed (all of the Lab Groups in the above example). This means that you can only become a member of the Lab Group if you are invited by the Lab Group Owner.

2. Accepting Requests (identified by the link “Apply to group”). This means that any user of Protocol Exchange can request to join this Lab Group, this application will be accepted or declined by the Lab Group Owner.

3. Open (identified by the link “Join group”). This means that any user of Protocol Exchange can join this group.

If you are a member of a Lab Group you can share protocols, or make a list of publications associated with the group by entering the DOIs for each article.

kurgan lab group.PNG

The Kurgan Lab, for example, has uploaded 1 protocol and listed 30 publications.

Most of the Lab Groups on our site are from Academic Institutions, but it is also possible for Commercial organisations to create a Lab Group and upload their protocols.

Two example of these are:

Targefect Transfection Group, Targeting Systems

biocision

When their protocols are listed, for example, on the Browse section, it is clearly marked that they are contributed by suppliers.

supplier and community.PNG

Another defining feature of the Lab Group is that you can upload a logo! The maximum allowed dimenstions are 200 X 100 pixels (width x height). I have made a list of the labgroups that have logos below; some of them are rather beautiful!

Sherwood’s Lab, Duke University

Sorger Lab, Harvard Systems Biology

Suga’s Lab (Hiroaki Suga), The University of Tokyo

Wehkamp Lab, IKP Stuttgart

Zaffran group, INSERM unit 910, Université de la Méditerranée

Guaza´s Lab, Cajal Institute, Madrid

Haseeb’s Lab (King Saud University, Riyadh, Saudi Arabia)

Kurgan Lab (University of Alberta)

Ren Lab, Oregon Health & Science University

Sanquin

Targefect Transfection Group, Targeting Systems

biocision

………………………………………………………..

More information about Lab Groups can be found in the About page.*

If you find that you have a protocol on the Exchange that was migrated from the old Protocols Network, and you want to get connected with the group that was automatically created, please do get in touch at: protocol.exchange@nature.com.

The arrival of guidelines for reporting experiments

Are you conducting experiments using animals or developing protocols in this area of research? If so, you may be interested to take a look at the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines when reporting your results and writing up those experimental procedures. These guidelines were published in June 2010 to improve the reporting of research using animals and can be found here. The guidelines themselves are presented in Table 2 of this paper, and consist of twenty items that can be used as a checklist for authors preparing a manuscript on this topic. Of course they are useful for peer-reviewers and journal editors too!

The ARRIVE guidelines provide recommendations about the content of all the standard sections of a manuscript, covering many points you would expect such as ethics statements and description of the experimental procedures and animals used. In addition, they suggest how to report the statistical analysis of results, as well as providing information about the study design, which is particularly relevant when writing up a protocol. To apply these guidelines to the preparation of a protocol for Nature Protocols, an Experimental Design section in the Introduction can be a good place to include further information on study design and the housing and husbandry of experimental animals, while the Procedure and Anticipated Results sections obviously allow detailed description of the methods used to obtain and analyse the results.

Although we do not publish primary research at Nature Protocols, but instead focus on tried-and-tested methods that have been previously published, we do find that these guidelines provide a useful resource and we encourage our authors to comply with the ARRIVE reporting guidelines when documenting protocols that involve animal studies.