Methods Digest, Vol 84, Issue 1

Nikola Loncar via methods%40net.bio.net (by hemicar085 from yahoo.com)
Tue May 1 12:41:17 EST 2012


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 hemicar085 from yahoo.com and I can send you some papers with detailed description of procedures. 


Kind regards, 
N.



________________________________
 From: "methods-request from oat.bio.indiana.edu" <methods-request from oat.bio.indiana.edu>
To: methods from magpie.bio.indiana.edu 
Sent: Tuesday, May 1, 2012 7:05 PM
Subject: Methods Digest, Vol 84, Issue 1
 
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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 <Gerchman from research.haifa.ac.il>
Subject: Trypsin and chemotrypsin colorimetric assay
To: <methods from oat.bio.indiana.edu>
Message-ID: <55e2515cbf7f576df6cccc68f469cd52 from 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.joshi from gmail.com>
Subject: Re: Ammonium sulfate precipitation from solutions with high
    sugar    content
To: WS <novalidaddress from nurfuerspam.de>
Cc: methods from magpie.bio.indiana.edu
Message-ID:
    <CAPaWRtgT-JLE0Hd2fdXVyWrjAWkYO1tYuowxdj5z_cVZUsdJvQ from 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 <novalidaddress from 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 from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>


------------------------------

Message: 3
Date: Mon, 30 Apr 2012 15:09:39 -0500
From: Michael Sullivan <mlsulliv from wisc.edu>
Subject: Re: Ammonium sulfate precipitation from solutions with high
    sugar    content
Cc: methods from magpie.bio.indiana.edu
Message-ID: <94BF9FAF-52D9-494A-B595-80E138E635FC from 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 from net.bio.net
> http://www.bio.net/biomail/listinfo/methods



------------------------------

Message: 4
Date: Mon, 30 Apr 2012 14:22:18 -0700 (PDT)
From: WS <novalidaddress from nurfuerspam.de>
Subject: Re: Ammonium sulfate precipitation from solutions with high
    sugar    content
To: bionet.molbio.methds-reagnts from googlegroups.com
Cc: methods from magpie.bio.indiana.edu, WS <novalidaddress from nurfuerspam.de>
Message-ID:
    <25407106.1986.1335820938409.JavaMail.geo-discussion-forums from 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 <novalidaddress from nurfuerspam.de>
Subject: Re: Ammonium sulfate precipitation from solutions with high
    sugar    content
To: methods from net.bio.net
Cc: methods from magpie.bio.indiana.edu, WS <novalidaddress from nurfuerspam.de>
Message-ID:
    <25407106.1986.1335820938409.JavaMail.geo-discussion-forums from 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 <theresahsu8 from live.com>
Subject: Membrane_protein_purification with_HIC
To: <methods from magpie.bio.indiana.edu>
Message-ID: <BLU151-W36010C37B2EEAE52F4268889280 from 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 <irappley from scripps.edu>
Subject: RE: Ammonium sulfate precipitation from solutions with high
    sugar    content
To: WS <novalidaddress from nurfuerspam.de>,
    "bionet.molbio.methds-reagnts from googlegroups.com"
    <bionet.molbio.methds-reagnts from googlegroups.com>
Cc: "methods from magpie.bio.indiana.edu" <methods from magpie.bio.indiana.edu>
Message-ID:
    <D87B51E608ECDD4583AF69B865ED2E73A196B2FCC7 from 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-bounces from oat.bio.indiana.edu [methods-bounces from oat.bio.indiana.edu] On Behalf Of WS [novalidaddress from nurfuerspam.de]
Sent: Monday, April 30, 2012 2:22 PM
To: bionet.molbio.methds-reagnts from googlegroups.com
Cc: methods from 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

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Methods mailing list
Methods from net.bio.net
http://www.bio.net/biomail/listinfo/methods



------------------------------

Message: 8
Date: Tue, 01 May 2012 11:13:32 -0400
From: AllisonH <nospam from gmail.com>
Subject: Re: Protein concentration with PEG
To: methods from net.bio.net
Message-ID: <x4Tnr.127056$O52.2469 from 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 <novalidaddress from nurfuerspam.de>
Subject: Re: Ammonium sulfate precipitation from solutions with high
    sugar    content
To: bionet.molbio.methds-reagnts from googlegroups.com
Cc: methods from magpie.bio.indiana.edu, WS <novalidaddress from nurfuerspam.de>
Message-ID:
    <12034566.332.1335871086444.JavaMail.geo-discussion-forums from 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 <novalidaddress from nurfuerspam.de>
Subject: Re: Ammonium sulfate precipitation from solutions with high
    sugar    content
To: methods from net.bio.net
Cc: methods from magpie.bio.indiana.edu, WS <novalidaddress from nurfuerspam.de>
Message-ID:
    <12034566.332.1335871086444.JavaMail.geo-discussion-forums from 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



------------------------------

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