Dear Yoram,
I would recommend p-nitroanilide substrate analogues assays since these are measured at 405 nm, they are extremely specific. For trypsin you would need benzoyl-arginine-p-nitroanilide and for chymotrypsin succinil-phenylalanine-p-nitroanilide. If you need more details, please contact me on hemicar...@yahoo.com and I can send you some papers with detailed description of procedures. Kind regards, N. ________________________________ From: "methods-requ...@oat.bio.indiana.edu" <methods-requ...@oat.bio.indiana.edu> To: meth...@magpie.bio.indiana.edu Sent: Tuesday, May 1, 2012 7:05 PM Subject: Methods Digest, Vol 84, Issue 1 Send Methods mailing list submissions to methods@net.bio.net To subscribe or unsubscribe via the World Wide Web, visit http://www.bio.net/biomail/listinfo/methods or, via email, send a message with subject or body 'help' to methods-requ...@net.bio.net You can reach the person managing the list at methods-ow...@net.bio.net When replying, please edit your Subject line so it is more specific than "Re: Contents of Methods digest..." Today's Topics: 1. Trypsin and chemotrypsin colorimetric assay (Gerchman) 2. Re: Ammonium sulfate precipitation from solutions with high sugar content (Pow Joshi) 3. Re: Ammonium sulfate precipitation from solutions with high sugar content (Michael Sullivan) 4. Re: Ammonium sulfate precipitation from solutions with high sugar content (WS) 5. Re: Ammonium sulfate precipitation from solutions with high sugar content (WS) 6. Membrane_protein_purification with_HIC (Theresa H) 7. RE: Ammonium sulfate precipitation from solutions with high sugar content (Irit Rappley) 8. Re: Protein concentration with PEG (AllisonH) 9. Re: Ammonium sulfate precipitation from solutions with high sugar content (WS) 10. Re: Ammonium sulfate precipitation from solutions with high sugar content (WS) ---------------------------------------------------------------------- Message: 1 Date: Mon, 30 Apr 2012 20:14:14 +0300 From: Gerchman <gerch...@research.haifa.ac.il> Subject: Trypsin and chemotrypsin colorimetric assay To: <meth...@oat.bio.indiana.edu> Message-ID: <55e2515cbf7f576df6cccc68f469c...@research.haifa.ac.il> Content-Type: text/plain; charset=UTF-8 Greetings netters I am looking for recommendations for Trypsin and Chemotrypsin colorimetric assay, preferably not in the UV range. Mant thanks Yoram ------------------------------ Message: 2 Date: Mon, 30 Apr 2012 14:18:49 -0400 From: Pow Joshi <pow.jo...@gmail.com> Subject: Re: Ammonium sulfate precipitation from solutions with high sugar content To: WS <novalidaddr...@nurfuerspam.de> Cc: meth...@magpie.bio.indiana.edu Message-ID: <capawrtgt-jle0hd2fdxvywrjawkyo1tyuowxdj5z_cvzusd...@mail.gmail.com> Content-Type: text/plain; charset=ISO-8859-1 Hi Wo, I was wondering if you could simply use the crude extract to spin down and create a sucrose density gradient...that will allow you to pick out some proteins within the gradient, and then the remaining may go over for ammonium sulphate precipitation ? ...I've no idea how this would work, but just a thought, if you have'nt already considered it. Pow On 30 April 2012 03:52, WS <novalidaddr...@nurfuerspam.de> wrote: > Dear Experts, > > I am attempting to precipitate small amounts of (total) protein from a > fruit juice concentrate. It looks quite much like honey. My plan is to > dilute it 1x with water (to reduce viscosity) and then saturate it > with ammonium sulfate. As the solution contains high amounts of sugars > (mostly saccharose, I assume), will these interfere with my > precipitation process? > > If yes, what may I do to circumvent it? Probably dialysis, but any > other ideas? > > Thanks for your help! > > Wo > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > ------------------------------ Message: 3 Date: Mon, 30 Apr 2012 15:09:39 -0500 From: Michael Sullivan <mlsul...@wisc.edu> Subject: Re: Ammonium sulfate precipitation from solutions with high sugar content Cc: meth...@magpie.bio.indiana.edu Message-ID: <94bf9faf-52d9-494a-b595-80e138e63...@wisc.edu> Content-Type: text/plain; CHARSET=US-ASCII What do you want to use the protein for? I had a very dilute, yet viscous extract of flowers that I wanted to use for western blotting. Carbohydrates made it visous, interfered with protein assays, and made the samples run quite poorly in a gel. I ended up using a phenol extraction method followed by MeOH precipitation that worked quite nicely. Of course, if you are trying to recover active protein this could be a problem. Mike --- Michael L. Sullivan, PhD Research Molecular Geneticist US Dairy Forage Research Center 1925 Linden Drive Madison, WI 53706 608-890-0046 (Phone) 608-890-0076 (FAX) On Apr 30, 2012, at 2:52 AM, WS wrote: > Dear Experts, > > I am attempting to precipitate small amounts of (total) protein from a > fruit juice concentrate. It looks quite much like honey. My plan is to > dilute it 1x with water (to reduce viscosity) and then saturate it > with ammonium sulfate. As the solution contains high amounts of sugars > (mostly saccharose, I assume), will these interfere with my > precipitation process? > > If yes, what may I do to circumvent it? Probably dialysis, but any > other ideas? > > Thanks for your help! > > Wo > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods ------------------------------ Message: 4 Date: Mon, 30 Apr 2012 14:22:18 -0700 (PDT) From: WS <novalidaddr...@nurfuerspam.de> Subject: Re: Ammonium sulfate precipitation from solutions with high sugar content To: bionet.molbio.methds-reag...@googlegroups.com Cc: meth...@magpie.bio.indiana.edu, WS <novalidaddr...@nurfuerspam.de> Message-ID: <25407106.1986.1335820938409.JavaMail.geo-discussion-forums@vbbgl4> Content-Type: text/plain; charset=ISO-8859-1 Hi Pow, long time no see :) My big problem is that there is almost no protein in the sample -> I won't see any bands. I expect about 1mg from 50 ml. I have started with 50ml of fruit juice concentrate, diluted with 50ml water and then added ammonium sulfate until saturation: No visible turbidity, No (visible) pellet after 2hrs @ 4000g (except some ammonium sulfate crystals. Unfortunately, I do not have access to a high speed centrifuge for such volumes. Spinning 24 eppis with 2ml each @ 15.000g in my small benchtop centrifuge might be an option, however. Could it make sense to add some unrelated protein, eg BSA or OVA (that's what I have in my fridge), as co-precipitant (like one does with glycogen or poly-acrylamide for DNA)?. Wo ------------------------------ Message: 5 Date: Mon, 30 Apr 2012 14:22:18 -0700 (PDT) From: WS <novalidaddr...@nurfuerspam.de> Subject: Re: Ammonium sulfate precipitation from solutions with high sugar content To: methods@net.bio.net Cc: meth...@magpie.bio.indiana.edu, WS <novalidaddr...@nurfuerspam.de> Message-ID: <25407106.1986.1335820938409.JavaMail.geo-discussion-forums@vbbgl4> Content-Type: text/plain; charset=ISO-8859-1 Hi Pow, long time no see :) My big problem is that there is almost no protein in the sample -> I won't see any bands. I expect about 1mg from 50 ml. I have started with 50ml of fruit juice concentrate, diluted with 50ml water and then added ammonium sulfate until saturation: No visible turbidity, No (visible) pellet after 2hrs @ 4000g (except some ammonium sulfate crystals. Unfortunately, I do not have access to a high speed centrifuge for such volumes. Spinning 24 eppis with 2ml each @ 15.000g in my small benchtop centrifuge might be an option, however. Could it make sense to add some unrelated protein, eg BSA or OVA (that's what I have in my fridge), as co-precipitant (like one does with glycogen or poly-acrylamide for DNA)?. Wo ------------------------------ Message: 6 Date: Mon, 30 Apr 2012 21:03:25 +0000 From: Theresa H <theresah...@live.com> Subject: Membrane_protein_purification with_HIC To: <meth...@magpie.bio.indiana.edu> Message-ID: <blu151-w36010c37b2eeae52f4268889...@phx.gbl> Dear all Can membrane proteins be purified with hydrophobic interaction chromatography (HIC) without denaturing/delipidate protein or the resin? Is there any type of detergents to avoid? Thank you. Theresa ------------------------------ Message: 7 Date: Mon, 30 Apr 2012 21:52:18 -0700 From: Irit Rappley <irapp...@scripps.edu> Subject: RE: Ammonium sulfate precipitation from solutions with high sugar content To: WS <novalidaddr...@nurfuerspam.de>, "bionet.molbio.methds-reag...@googlegroups.com" <bionet.molbio.methds-reag...@googlegroups.com> Cc: "meth...@magpie.bio.indiana.edu" <meth...@magpie.bio.indiana.edu> Message-ID: <d87b51e608ecdd4583af69b865ed2e73a196b2f...@exch-ccr01.lj.ad.scripps.edu> Content-Type: text/plain; charset="us-ascii" Hi Wo, In the past I've added 1 mg/mL BSA to a dilute protein solution, then precipitated it with TCA. However, then you'll be stuck with 1 mg/mL BSA in your resulting pellet. If you're seriously considering the 24 eppis option, wouldn't dialysis be easier? HTH, Irit _______________________________________ From: methods-boun...@oat.bio.indiana.edu [methods-boun...@oat.bio.indiana.edu] On Behalf Of WS [novalidaddr...@nurfuerspam.de] Sent: Monday, April 30, 2012 2:22 PM To: bionet.molbio.methds-reag...@googlegroups.com Cc: meth...@magpie.bio.indiana.edu; WS Subject: Re: Ammonium sulfate precipitation from solutions with high sugar content Hi Pow, long time no see :) My big problem is that there is almost no protein in the sample -> I won't see any bands. I expect about 1mg from 50 ml. I have started with 50ml of fruit juice concentrate, diluted with 50ml water and then added ammonium sulfate until saturation: No visible turbidity, No (visible) pellet after 2hrs @ 4000g (except some ammonium sulfate crystals. Unfortunately, I do not have access to a high speed centrifuge for such volumes. Spinning 24 eppis with 2ml each @ 15.000g in my small benchtop centrifuge might be an option, however. Could it make sense to add some unrelated protein, eg BSA or OVA (that's what I have in my fridge), as co-precipitant (like one does with glycogen or poly-acrylamide for DNA)?. Wo _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods ------------------------------ Message: 8 Date: Tue, 01 May 2012 11:13:32 -0400 From: AllisonH <nos...@gmail.com> Subject: Re: Protein concentration with PEG To: methods@net.bio.net Message-ID: <x4Tnr.127056$o52.2...@unlimited.newshosting.com> Content-Type: text/plain; charset=ISO-8859-1; format=flowed How about Ficoll? The powder can be used just like PEG. Allison On 30/04/2012 7:50 AM, WS wrote: > Dear Experts, > > You are probably aware of the protein concentration method by > "dialyzing" against polyethyleneglycol. My question is regarding the > MW of PEG and the pore size of the tubing: Currently, I am using > PEG20000 (avg MW 15k-20k) and a dialysis tubing with a cutoff of 14kD > for globular proteins. In my experience, this is working very well > (and cheap), esp. for large volumes. > > We just got some concerns now that some of the PEG could make it > across the membrane. Is there any need to be concerned? Which would > mean to switch to PEG35k or higher and/or tubing with a lower cut/off, > both increasing the expenses. As I understand PEG, the molecules will > get quite huge due to hydratation and PEG always has some low MW size > "contaminants" i.e. molecules shorter than desired. > > Anything you can suggest? > > Thanks! > > Wo ------------------------------ Message: 9 Date: Tue, 1 May 2012 04:18:06 -0700 (PDT) From: WS <novalidaddr...@nurfuerspam.de> Subject: Re: Ammonium sulfate precipitation from solutions with high sugar content To: bionet.molbio.methds-reag...@googlegroups.com Cc: meth...@magpie.bio.indiana.edu, WS <novalidaddr...@nurfuerspam.de> Message-ID: <12034566.332.1335871086444.JavaMail.geo-discussion-forums@vbpz13> Content-Type: text/plain; charset=ISO-8859-1 Hi Michael, I do not need active protein, I need it for a western. I'll give it a try, thanks! Wo ------------------------------ Message: 10 Date: Tue, 1 May 2012 04:18:06 -0700 (PDT) From: WS <novalidaddr...@nurfuerspam.de> Subject: Re: Ammonium sulfate precipitation from solutions with high sugar content To: methods@net.bio.net Cc: meth...@magpie.bio.indiana.edu, WS <novalidaddr...@nurfuerspam.de> Message-ID: <12034566.332.1335871086444.JavaMail.geo-discussion-forums@vbpz13> Content-Type: text/plain; charset=ISO-8859-1 Hi Michael, I do not need active protein, I need it for a western. I'll give it a try, thanks! Wo ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 84, Issue 1 ************************************** _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
