Jim Hardy › Comments

No luck. More direct link here: http://www.sciencedirect.com/science... - Heather

@Heather, thanks for the direct link. @Jim, check yer email :-) - Graham Steel

Thanks to Abhishek, who also sent it to me! So if Jim, you didn't get it through Graham or Abhishek... drop a line. - Heather

?? That IS the full article..... - Graham Steel

Yes, I know. Looking for a PDF. HTML is good for some things, but…. - Jim Hardy from email

Seems there is no official PDF version. Printing the page as a PDF is an option. - Pedro Matos

But I wonder how they're going to generate the biomass, first in terms of feedstock (algae). I am also skeptical of any vegetarian who is trying to make "artificial" meat. Soilent Green!! - Jim Hardy

"It couldn't be more unnatural..." - well as described, yes, but not all meat is high-intensity, factory-farmed. Or don't they have free-range in the USA? - Neil Saunders

Also a bit skeptical to the promise of this technology to replace meat-production. Nice if this can eliminate vegetarians ;-) also like the possibility of eliminating the bacterial contamination risk... - Nils Reinton

@Jim - assuming your comment wasn't tongue-in-cheek, why would you be skeptical? many vegetarians are so because they object to the way the animals are treated. treatment which, in many cases, makes them much less healthy to eat - which is another reason behind vegetarianism. so why wouldn't a vegetarian be just as capable as an omnivore to drive this technology? (OTH if you meant your comment purely to set up the Soylent Green punchline, than I apologize - that did make me chuckle ;-) - tim

@Nils other than the objections raised in the article, which are formidable, what makes you skeptical? people won't eat it if they know it's in vitro? political will to revamp the food industry isn't there? or some technological weakness? - tim

@tim in addition to your points on political will and consumer skepticism, it seems to me that there are huge technological obstacles (like vascularization also mentioned in the article). Lastly, I don't think traditional meat producers are just happily going to roll over and die. - Nils Reinton

"Lastly, I don't think traditional meat producers are just happily going to roll over and die". Quite. Slightly OT, but cue 'Soylent Green' featuring Heston C et al - http://www.youtube.com/watch... - Graham Steel

@Nils I agree. but if they can sell it to the food industry - cheaper to produce, safer, smooth transition to new pipeline, profits to be had...well, call me idealistic but I still have a shred of hope. (technology seems to me to be a smaller hurdle, but maybe I'm just jaded.) - tim

Depends a bit on the yield of recombinant. Has it been through ion-exchange (e.g. miniQ) and gel filtration (e.g. S200/S75, depends on size) already? Those are the usual first steps for non-tagged. - Neil Saunders

This is something I'm buying at 4mg/ml, 90% pure -- meaning, I presume, that 0.4mg/ml is E. coli crap and 3.6 mg/ml is my recombinant. I don't know what purification steps have already been used -- I am trying to track that info down -- my guess would be that it's just the usual suspects -- ammonium sulfate cut, size exclusion, HPLC (RP or ion). That's why I'm thinking in terms of home-made affinity columns as above. - Bill Hooker

And no, for various reasons I can't just buy a better product! :-) - Bill Hooker

At this point (assuming size, pI, etc has been tried), it sounds like you'd be better off cooking up a chromatography step based on affinity to the recombinant (e.g. substrate analog, etc) rather than broad immunoprecip. towards the contaminants. - Wladimir Labeikovsky

Wladimir, the protein has no activity so there's no substrate -- I could use an antibody, but why do you think it would be better to try to recover the 90% target than to try to yank out the 10% contaminant? I wasn't thinking of straight immunoprecip, rather a protein G column + polyclonal + crosslinking = affinity column for the contaminants, e.g. http://cshprotocols.cshlp.org/cgi... - Bill Hooker

I should add that I'll be trying to clean up 20-50 mg at a time, if I can scale up that far. That's why I thought to focus on the E. coli crap -- less to bind. - Bill Hooker

My first step would be to run a gel on it. You may find that that 10% isn't even protein but salt and nucleic acids. Then I would try in order: Size exclusion, ion exchange, hydrophobic interaction chromatography to see if they help you clean it up or not. SEC probably not so good on a 20mg scale but the others can be done in batch mode in good old Buchner funnels. - Cameron Neylon

I have some gels run by my "predecessors" on earlier batches -- they do show faint Coomassie stainable bands in addition to the strong band of my target protein. There may be some NA but at least some of the crud is protein. The reason the crud is a problem is that this is to be used to assay for anti-(target protein) antibodies in human serum, and 10% of such serum contains antibodies against various E. coli proteins, which gives a false positive readout in the assay. - Bill Hooker

As others have said, I'd run an ion exchange first (mainly because it's fast) and see how far that gets you, then move to a size-exclusion column if you think you have enough mass separation. You could also consider running a quick mass spec or something similar on the supplied material to get a better handle on the impurities. - Jason Winget

the protein G column is worth trying if you have it set up already but there's no guarantee at all that it would catch the particular contaminants you have - Wladimir Labeikovsky

Adding my voice to the rest - column sep is the way to go at first, just to get a feel for it. - Mr. Gunn

@MrG, do you mean ion exchange? And can anyone recommend a good resin for that? (I have a peristaltic pump but I don't know if it's in good working order...) - Bill Hooker

At pH > pI, protein net charge (-), anion exchange (e.g. mini Q). pH < pI, protein net charge (+), cation exchange (e.g. mini S). Also recommend a friend with an FPLC setup :-) - Neil Saunders

Oh yeah -- I don't know *exactly* what the recombinant is. Some genius stuck me with this; it's a commercial product, one manufacturer and one distributor (who know they have us over a barrel) and so all I know is that it "encompasses a portion" of a particular protein. So I can't even predict pI from sequence. We're skint so HPLC/FPLC is out of the question... I know people with rigs but I'm loath to impose to the necessary degree (testing, optimisation, scale-up...). - Bill Hooker

Hmm, problematic. So I guess SDS-PAGE (as others suggested) will give you monomeric size and so an indication of the best size exclusion column. Can you do 2D-PAGE to estimate pI? Presumably your protein will be the "major spot". Option 3: see where it comes down in ammonium sulphate cut and from there, hydrophobic interaction column. - Neil Saunders

As above, I have SDS-PAGE images -- and only now I realize, the apparent size tells me that it's most if not all of the protein in question. So I *can* guesstimate pI from sequence.... it's just over 5, most likely. So at a nice physiological 7.4, anion exchange it is. If I go that way. Why are y'all bastards so against my proteinG+polyclonal idea? - Bill Hooker

My worry would be that a polyclonal for all E.coli could (a) miss some and (b) cross-react with the recombinant. - Neil Saunders

Ah, crap -- important point that I thought I had put in the first couple of comments: the polyclonal I propose to use is the one that is also used in an EIA to measure the degree of E. coli contamination in the antigen. When the EIA reactivity gets below a certain threshold, the antigen is useable in the product for which it's intended. So I can be confident the polyclonal won't miss anything I care about or pull out the recombinant. - Bill Hooker

Note that all of my info comes from fragmentary notes left behind by my predecessors, so some/all of it might be garbage. So I'm grateful not only for criticism of the polyclonal idea, but also for all the various alternatives being put forward. I need to solve this problem pronto so I think I will run at least two approaches in parallel. As of now I'm leaning towards Mini-Q and the polyclonal thingy, the latter probably as a simple batch IP initially and then on a column if that isn't good enough. - Bill Hooker

Isn't using the same using the same polyclonal produce sort of a circular argument for quality? Also, the anecdotal experience in my former lab was that sera that performed well in one application may not work well under another application with different conditions. That said, it looks like you're in a pickle, and the pull-down approach may be your best bet. - Brian Haugen

"...produce sort of a circular argument for quality?" -- yes, it does rather. And also yes, my experience has been that sera don't always work well in multiple applications. The saving grace is that I don't have to clean this stuff up *extensively* so the polyclonal thing might work. I *think* it's how the cleanup was done previously... as I mentioned, I have only fragmentary records to work from. - Bill Hooker

Muchas, muchas gracias for all the input, folks. (And keep the ideas coming, if any more occur to you!) - Bill Hooker

Bill, if you have a polyclonal "scavenger" you can buy just buy some S Avidin beads and biotinylate the Ab. It's pretty simple and could be done in "batch" mode in buchner funnel, as Cameron suggested. Also, activated resin from Thermo http://is.gd/2FYzg (and others) allows you to link you Ab to resin and would work the same way. SA + Biotinylated Ab would be the old school way of doing it. - Jim Hardy

Jim, would streptavidin/biotin be better than simply using proteinA/G beads with the unmodified Ab? - Bill Hooker

prizes ;) - D0r0th34

Exactly. But what for? - Graham Steel

Courage? http://blindscientist.genedrift.org/2008... - Paulo Nuin

send them to people who research blog an article from one of the plos journals? Or just people who ask politely? :D - Allyson Lister

Nice one, Allyson. Will run this by Dave Munger of researchblogging.org - Graham Steel

And have now done so. - Graham Steel

Me! Me! Me! Want! Want! Want! (subtle, huh?) :) - Ricardo Vidal

Wore mine from #sbcPA yesterday. All the non-science peeps needed explainations. - Jim Hardy

@ Ricardo, (you is as subtle as a brick shaped object) what size are you? I'll trade you one for a Mendeley one :) - Graham Steel

Since I've yet to hear back from Dave M, plan T is now underway. Here are the shirts http://www.flickr.com/photos... Top row middle is now winging it's way off to Dr H Gee of Nature.com http://twitter.com/McDawg... even though, this might be rejected via i-stone !!. Ricardo V (shirt size yet to be disclosed) might have another (he has 12 hours left to comment) , so there are still four avaliable for free from me as matters stand. Code = ask politely/candidly. #PLOS - Graham Steel

After what I suffered in the link posted above, I think I would be extremely happy to have a new PLoS T. Thanks! - Paulo Nuin

Paulo, one can still do S, or L or XL. (One XL one went to someone in Cromer). Name your size and it will be shipped. ONLY three left now and you folks must try harder to convince me give this stuff away, err convincingly. (Each pack will contain other PLoS goodies too). - Graham Steel

I will take the L. Can you send me an email with whatever you need? Maybe it's too far for you to ship :-) - Paulo Nuin

And the L will be shipped to Paulo later in the month. So what's left? After a re-count, 2 S and 1 XL. Rather than simply giving the last 3 away in the manner thus far, one shall devise a competition and three lucky winners will get the last of the pickings. - Graham Steel

Hmmm - somehow this comment thread dropped off my first few FF pages... Good luck with the competition, I might be a M but I'm not a S, so I'll pass :) - Allyson Lister

Competition Idea ! As matters stand, me thinks it's boiling down to some form of pub quiz thang down in the, err pub after Science Online London http://www.scienceonlinelondon.org/blog... It will last for 5 - 10 mins and will be called "Who's Round Is It Anyway" Whilst the rules will be made up as we go along, the Host (not me) will ensure 'fair-play'. Cheating will be allowed within reason. 10 Prizes up for grabs inc. a priceless mystery top prize !! #solo09 - Graham Steel

Above comment updated, nudge. - Graham Steel

Graham - I'll happily take you up on the trade-for-Mendeley shirt! :-) Yes yes yes! - Victor / Mendeley Team

Game on. Must dash - I have a train to catch...whooosh - Graham Steel

I recently posted on researchblogging.org and have published in PLoS ONE. Does that qualify me for one of the red or black ones in size M? - Karen James

Did I read Mendeley shirt? (am actually wearing one of my PLoS One shirts right now...) - Björn Brembs

I'm debating whether to apply for enrollment. Obviously, the open aspect of it is very cool. But this isn't kidding around - medical history and personal information become public domain (CC-0!): http://www.personalgenomes.org/public.... It's not an extension of yourself going open (i.e. your research), it's _you_... and I need to think a little more about the implications. - Shirley Wu

Yeah, it's why I haven't done it yet. Just haven't had the time to think through the implications - Deepak Singh

But your name will not end up in the CC0 data, will it? Why should I care if my digital copy ends up in that data set if no one would be able to link that to me? Got to read the ToS right now... - Egon Willighagen

I suspect that the level of detail of information allows one to infer who it may be. Specially medical information. Also, from what I've seen, they take head shots of each person (with a little measuring tape on the forehead for scale!) :) - Ricardo Vidal

I signed up for more info in the first phase, but have been sitting on my invite. As cool as it would be, I'm not sure the risks outweigh the rewards, at least until better protections are put into place. GINA is a start, but there's much work to be done. - Chris Miller

Egon, I've spoken to enough people who say that you don't need a name with whole genome data to figure out whom it belongs to. I will likely end up doing it since I don't care even if they had my name up there, but just from the conceptual standpoint, it's good to understand. - Deepak Singh

Right, there is definitely more than enough information to connect you to your data. In fact, just the zip code, sex, and DOB are enough to identify 87% of people in a de-identified database (saw this stat yesterday, can't remember where), and for PGP you've got a fairly complete medical history as well as photos. I'm pretty torn about this because I really think a large, open database... more... - Shirley Wu

You also have to worry about the privacy of your kids, I think, even if they're not born yet...very frustrating and confusing issue! - Steve Koch

I signed up months ago and have passed the first qualifying tests. On to level two, for consideration as one of the PGP100 or PGP1000 if I don't make the first cut. I have no fears of my DNA being exposed. - Jim Hardy

@Jim, I assume that means you also have no problem with people knowing your medical history, including, hypothetically, potentially "too much information" about, say, that anal fissure you had a year ago or your chronic irritable bowel syndrome? - Shirley Wu

Agreed. Facebook is for socializing, family, friends. FriendFeed, twitter used, although not exclusively, for science and work stuff. - Jim Hardy

+1 to Garret, Egon and Jim - exactly how I divide it :) - Allyson Lister

Facebook = Friendbook, FriendFeed = WorkFeed. All nicely partitioned until merger made worlds collide. - Richard Akerman

Are we gonna have "death panels" here? - Paulo Nuin

Obama-who ?? Care-what ?? I'm from a socialist country remember. We're used to death panels apparently - bring it on .... - Nils Reinton

Socialism!! - Paulo Nuin

oh dear.. - Frank

"Like" as in "hmm, interesting..." - Neil Saunders

+1 Frank - Allyson Lister

great approach, one of companies that I'm watching - Alexey

I just heard their CTO speak at the San Diego Biotech Network meeting today. Got permission to blog about it and talked to him for a while about my dissertation, which was, as it happens, on the use of small molecules to manipulate adult stem cell differentiation. - Mr. Gunn

@Mr.Gunn - apply http://www.fatetherapeutics.com/careers... you may fit :) - Alexey

Alexey, he asked me to forward my resume to him, so hopefully he'll followup if there's real interest. There's probably few people in the world who are a better fit than me for one of their positions. - Mr. Gunn

Scientists are humans, too. We need to keep reminding ourselves of it. Like the Romans, where the guy holding the laurel wreath for the successful general during the victory parade through Rome was always repeating : "remember, you're only human". - Björn Brembs

Mandy Rice-Davies applies, but I would argue that the solution is to show your data. You are unlikely to slide down the peer-->maverick-->pariah slope if your data are good and your reasoning sound; the most anyone will do is disagree with you, and say why, but they will still respect you. - Bill Hooker

so industry does look to academia postdoc salary as a guide? That kinda seems wrong. I mean, academia isn't appreciated for the pay, but you do have more autonomy. - Mr. Gunn

I don't know that they look to academic salaries as a guide, they just know perfectly well that at a salary they consider a bargain, the postdoc is still getting a huge raise compared to academia. Perhaps the better question is: does industry have a postdoc trap too? Is it possible to dead-end the way postdocs usually do in academia? - Bill Hooker

Bill, that's kinda what I was asking. I had the first interview of what's apparently quite a process today for a postdoc with GNF. http://gnf.org While they said they were very interested in the professional development of people in this position, when I asked what kinds of institution-sponsored professional development things were available, they couldn't tell me much. The most... more... - Mr. Gunn

Andrew Su (http://friendfeed.com/asu) works at GNF -- I'm sure he wouldn't mind if you sent him a few questions by email. He's on LinkedIn so maybe you have a connection, too. - Bill Hooker

2 handed freedom still couple days away :( - Attila Csordas

Liking for the fact that you're (nearly) healed, not the accident! - Bill Hooker