protein dimerization in SDS-PAGE
Jayakumar, R
I have this small 15 kDa protein. I keep getting dimerization of this protein in SDS-PAGE. I have tried Beta-ME, DTT etc. in different concentrations and all that in the SDS sample buffer. But still I get these dimers of the same protein. Sometimes the monomer altogether disappears with appearance of a higher molecular weight dimer. I know that treatment with some compounds like iodoacetamide prevents such problems. But I dont know a good working protocol which is easy and fast. I dont want to do any dialysis either for certain reasons. I really appreciate any help given.
Thanks everyone in advance.
bye
Jayakumar
---
Jayakumar, R
Thank you both for your help. Let me try it out this week. But do you have any idea whether an alkylating agent like iodoacetamide (IAM) would have any effect on this. I heard that sometimes the reduced disulphide groups tend to get oxidized again in the stacking part of the gel due to ammonium persulphate and this can be prevented if the cysteins in the protein is irreversibly alkylated by IAM. I am trying to design a step using IAM after the sample buffer heating step.
Do you know anything about this?
thank you
Jayakumar
> ----------
> From: Michael
> Sent: August 8, 2003 11:56 AM
> To: Jayakumar, R
> Subject: Re: protein dimerization in SDS-PAGE
>
> <<File: kolbe.vcf>>
> Hi Jayakumar,
> just use 60 to 70 °C insteda of boiling via sample incubation with SDS-buffer. This is a problem which occurs frequently with hydrophobic proteins. I hope that will help you
> Cheers,
> Michael
---
Tom Anderson
> - Substitute dodecyl for lauryl sulfate everywhere.
Are dodecyl sulphate and lauryl sulphate not the same thing?
tom
--
So the moon is approximately 24 toasters from Scunthorpe.
Wolfgang Schechinger
In that case, some BME / DTT in the upper buffer (and pre-running the gel
without sample) might remove remains of APS.
Wo
Jayakumar, R
Jayakumar
> ----------
> From: D.K.
> Sent: August 9, 2003 12:40 AM
> To: met...@hgmp.mrc.ac.uk
> Subject: RE: protein dimerization in SDS-PAGE
>
> In article <97101976F8A044468CA7...@VISTA.roswellpark.org>, R.Jay...@RoswellPark.org ("Jayakumar, R") wrote:
> >Dear Michael and DK
> >Thank you both for your help. Let me try it out this week. But do you =
> >have any idea whether an alkylating agent like iodoacetamide (IAM) would =
> >have any effect on this. I heard that sometimes the reduced disulphide =
> >groups tend to get oxidized again in the stacking part of the gel due to =
> >ammonium persulphate and this can be prevented if the cysteins in the =
> >protein is irreversibly alkylated by IAM. I am trying to design a step =
> >using IAM after the sample buffer heating step. =20
> > Do you know anything about this? =20
> >thank you=20
> >Jayakumar
>
> Just IMHO: pretty unlikely that alkylating cysteines will change anything.
> In the pre-minigels era it used to be a standard part of sample
> preparation but now hardly anyone ever uses it for a routine gels.
> I'm right now working with protein that has 7 exposed cysteines
> and 100% of it runs as a monomer (even though I frequently
> use 2X more APS to make the gel polymerize faster).
>
> It is usually very hydrophobic proteins that form dimers/multimers
> in SDS-PAGE. Basically, they are so hydrophobic "inside" that
> the unfolded chains aggregate even in the presense of SDS (excluding
> it). So, you get paradoxal situtuaon - the more you denature these
> proteins, the more complexes are formed. Hence, don't try to unfold
> them completely - use milder detergent and RT instead of boiling.
>
> DK
>
> >> ----------
> >> From: Michael
> >> Sent: August 8, 2003 11:56 AM
> >> To: Jayakumar, R
> >> Subject: Re: protein dimerization in SDS-PAGE
> >>=20
> >> <<File: kolbe.vcf>>
> >> Hi Jayakumar,
> >> just use 60 to 70 =B0C insteda of boiling via sample incubation with =
> >SDS-buffer. This is a problem which occurs frequently with hydrophobic =
> >proteins. I hope that will help you
> >> Cheers,
> >> Michael
> >>=20
> >> "Jayakumar, R" wrote:
> >>=20
> >> > HI.. all
> >> > I have this small 15 kDa protein. I keep getting dimerization =
> >of this protein in SDS-PAGE. I have tried Beta-ME, DTT etc. in =
> >different concentrations and all that in the SDS sample buffer. But =
> >still I get these dimers of the same protein. Sometimes the monomer =
> >altogether disappears with appearance of a higher molecular weight =
> >dimer. I know that treatment with some compounds like iodoacetamide =
> >prevents such problems. But I dont know a good working protocol which =
> >is easy and fast. I dont want to do any dialysis either for certain =
Tom Anderson
> In article <Pine.LNX.4.44.030810...@crow.linux.ox.ac.uk>, Tom Anderson <univ...@herald.ox.ac.uk> wrote:
> >On Fri, 8 Aug 2003, D.K. wrote:
> >
> >> - Substitute dodecyl for lauryl sulfate everywhere.
> >
> >Are dodecyl sulphate and lauryl sulphate not the same thing?
>
> Oh, yes, indeed it is the same. Sorry - my mistake!
>
> But now I am thinking what exactly it was that I did about 10 years
> ago... It was bought as lauryl (and that's where my confusion came
> from). Umm, now I think it was lithium lauryl/dodecyl sulfate... No idea
> why it a different salt would make any difference (but I know it did).
As i understand it, the lithium form is more soluble than the sodium form;
i suppose it has something to do with the physical properties of the ions,
but it's been a long time since i did my A-levels!
If that's true, then presumably, a solution of LDS and NaCl is no better
than SDS, as the sodium ions from the salt will precipitate the dodecyl
sulphate. There isn't usually NaCl in the sample buffer itself, but i
can't remember off the top of my head what's in the running buffer: if
it's got Na+ ions, using LDS may not be helpful.
tom
--
REMOVE AND DESTROY
EK
>
> If that's true, then presumably, a solution of LDS and NaCl is no better
> than SDS, as the sodium ions from the salt will precipitate the dodecyl
> sulphate. There isn't usually NaCl in the sample buffer itself, but i
> can't remember off the top of my head what's in the running buffer: if
> it's got Na+ ions, using LDS may not be helpful.
>
> tom
No, no, Tom. It is potassium that will cause precipitation. Sodium is OK.
Moreover, even traces of potassium added to sodium DS will cause
precipitation.
-Emir
Jayakumar, R
thanks for all those tips pouring in . By the way, It should be lithium dodecyl sulphate. They use that to run low temperatures PAGES. LDS does not precipitate at low temperatures like SDS does.
Anyway, thanks for all that tips. Keep it coming. I am building up a new experiment now.
jayakumar
> ----------
> From: D.K.
> Sent: August 10, 2003 11:02 PM
> To: met...@hgmp.mrc.ac.uk
> Subject: Re: protein dimerization in SDS-PAGE
>
> In article <Pine.LNX.4.44.030810...@crow.linux.ox.ac.uk>, Tom Anderson <univ...@herald.ox.ac.uk> wrote:
> >On Fri, 8 Aug 2003, D.K. wrote:
> >
> >> - Substitute dodecyl for lauryl sulfate everywhere.
> >
> >Are dodecyl sulphate and lauryl sulphate not the same thing?
>
> Oh, yes, indeed it is the same. Sorry - my mistake!
>
> But now I am thinking what exactly it was that I did about 10 years
> ago... It was bought as lauryl (and that's where my confusion came
> from). Umm, now I think it was lithium lauryl/dodecyl sulfate... No idea
> why it a different salt would make any difference (but I know it did).
>
> DK
>
>
>
>
---
Tom Anderson
> > If that's true, then presumably, a solution of LDS and NaCl is no better
> > than SDS, as the sodium ions from the salt will precipitate the dodecyl
> > sulphate. There isn't usually NaCl in the sample buffer itself, but i
> > can't remember off the top of my head what's in the running buffer: if
> > it's got Na+ ions, using LDS may not be helpful.
>
> No, no, Tom. It is potassium that will cause precipitation. Sodium is
> OK. Moreover, even traces of potassium added to sodium DS will cause
> precipitation.
At low temperatures, SDS precipitates somewhat, but LDS doesn't - that's
what i was talking about. You are of course quite right that potassium
ions precipitate DS very effectively, and indeed, the precipitation of SDS
is a relatively minor effect. I am probably getting overexcited about the
whole matter.
tom
--
Space Travel is Another Word for Love!
Nick Theodorakis
SDS can ppt with very high concentrations of sodium, as well. In the
old days, we used to make binding buffer for oligo-dT columns with Li
instead of Na because it was more soluble under those conditions (0.5M
salt, 1% SDS).
Nick
--
Nick Theodorakis
nicholas_t...@urmc.rochester.edu
Stefan
could try 20mM NEM treatment of your samples prior to running them, 5
min on ice is fine. I've used this when looking specifically at SH vs
S-S bonds in non-reducing gels; the NEM reacts with all the free SH
groups to prevent further S-S formation.
Kyle Legate
Dr Engelbert Buxbaum
> - Substitute dodecyl for lauryl sulfate everywhere.
Lauryl is the trivial name for dodecyl. You may mean sodium tetradecyl
sulfate, which indeed can change electrophoretic behaviour of some
proteins (there were papers on this detergent and Na/K-ATPase in the
late '80s iirc).