Author Topic: Pellet solubilization after TCA/acetone?  (Read 22788 times)

Allison Mackay

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Pellet solubilization after TCA/acetone?
« on: March 29, 2011, 06:52:16 AM »
So I've been having a persistent problem after using TCA/acetone to precipitate serum protein (usually after Proteominer or if given samples in an SDS buffer).  Even if I am careful not to overdry the pellet, it forms a jelly pellet in the lysis buffer and is often quite resistant to resolubilizing.

Is there a secret trick I'm missing here?  Other than "vortex, sonicate, pipet, repeat until dissolved" which is my current method.

I think that going back to 2-D Cleanup kit might mitigate this, but we've stopped using it due to the added cost per sample.

Sjouke Hoving

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Re: Pellet solubilization after TCA/acetone?
« Reply #1 on: March 29, 2011, 07:37:24 AM »
You can try and use a small glass-teflon homogenizer, there are these very small ones. Otherwise, I would recommend to stay with the Clean up Kit.

sjouke

Allison Mackay

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Re: Pellet solubilization after TCA/acetone?
« Reply #2 on: March 29, 2011, 08:25:52 AM »
Thanks, I think we have the blue plastic ones that fit in an eppendorf tube as well.  I might try that.

dfried

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Re: Pellet solubilization after TCA/acetone?
« Reply #3 on: March 29, 2011, 09:16:39 AM »
I've also found fewer problems when ppt'ing only enough for each gel, rather than ppt'ing the whole thing and then resolubilizing the whole pellet.  We use a methanol/chloroform method.  You mentioned doing this out of SDS sample buffer, could the glycerol be causing a problem?

David

Allison Mackay

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Re: Pellet solubilization after TCA/acetone?
« Reply #4 on: March 29, 2011, 12:45:16 PM »
It's not standard SDS buffer for Western gels.  It's the 4% SDS/25 mM DTT buffer for eluting from Proteominer using the modified protocol of Di Girolama et al. 2011.

yudohaoxiu

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Re: Pellet solubilization after TCA/acetone?
« Reply #5 on: March 31, 2011, 09:52:29 AM »
I have the same problem. I use Perfect fucus purification kit. At the end of percipitation I use rehydration buffer (8 M Urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer) to redisolve the protein pellet .  I have also found the jelly pellet that difficulte to disolve back. People in GE suggest me to disolve it in room temperature for a while instead of putting it in ice, it seems to be working.
« Last Edit: April 01, 2011, 03:32:03 AM by yudohaoxiu »

Sjouke Hoving

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Re: Pellet solubilization after TCA/acetone?
« Reply #6 on: April 01, 2011, 02:46:22 AM »
People don't have to worry about working at room temperature with their protein sample, once it is in the strongly denaturing 2D lysis buffer.

sjouke

Allison Mackay

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Re: Pellet solubilization after TCA/acetone?
« Reply #7 on: May 05, 2011, 06:43:33 AM »
I've also found fewer problems when ppt'ing only enough for each gel, rather than ppt'ing the whole thing and then resolubilizing the whole pellet.

So I think that this may actually be the culprit!  More protein = overly large pellet = resists breaking up.  My current samples should have a lot of protein and the pellets are quite big.  That doesn't explain why physically mashing up the pellet doesn't help but maybe once it's formed a jelly mass it's going to remain resistant.  I may end up trying to add some SDS again, get it into solution, and retry the cleanup with the 2-D Cleanup kit (only doing part of the samples so the volume is smaller = uses less precipitant and gives a smaller pellet)

I'm wondering if the the "wash additive" in the 2-D cleanup kit somehow assists in getting the pellet back into solution afterward?  Or is it the composition of the wash solution itself?  The pellet has a very different consistency than that from TCA/acetone (which 2-D Cleanup appears to be based on).

mysg

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Re: Pellet solubilization after TCA/acetone?
« Reply #8 on: May 05, 2011, 07:56:04 AM »
I've also found fewer problems when ppt'ing only enough for each gel, rather than ppt'ing the whole thing and then resolubilizing the whole pellet.

So I think that this may actually be the culprit!  More protein = overly large pellet = resists breaking up.  My current samples should have a lot of protein and the pellets are quite big.  That doesn't explain why physically mashing up the pellet doesn't help but maybe once it's formed a jelly mass it's going to remain resistant.  I may end up trying to add some SDS again, get it into solution, and retry the cleanup with the 2-D Cleanup kit (only doing part of the samples so the volume is smaller = uses less precipitant and gives a smaller pellet)

I'm wondering if the the "wash additive" in the 2-D cleanup kit somehow assists in getting the pellet back into solution afterward?  Or is it the composition of the wash solution itself?  The pellet has a very different consistency than that from TCA/acetone (which 2-D Cleanup appears to be based on).

I use Biorad 2D clean up so the things might be really off, but I see very good size pellet even from small amount of lysate. I suspect the kit probably have some carrier that helps to form the pellet. When I told my friend to use this kit, my friend was reluctant because his past experience with ppt was terrible, but after using this kit, ppt became his favorite, because of this good visible pellet. This pellet or carrier we presume, is hard to dissolve once it is completely dry.  That's why we keep just tiny amount of acetone (<1mm in epp tube), and then directly disolve in 7M urea/2M thiourea/4% CHAPS buffer (at room temperature). This protocol seems working very well.

roy_arnab

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Re: Pellet solubilization after TCA/acetone?
« Reply #9 on: May 12, 2011, 04:08:58 AM »
Hello guys! i just wanted to add my experience here. I also had this problem of hard to dissolve ppt after 2D clean up or TCA/Acetone precipitation. It got solved when I started adding more Pharmalyte (~1%). Previously I used to add 0.5%. The ppt now dissolves promptly. Thanks to Hoving.

Allison Mackay

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Re: Pellet solubilization after TCA/acetone?
« Reply #10 on: July 21, 2011, 09:02:45 AM »
As the say on the interwebs: "BUMP".

I'm bringing this topic back to life because although I've shelved this problem for quite a while, I'd like to get these troublesome samples run and done.  So I need to solubilize those darn jelly pellets.

I had no luck with the in-tube (blue pestle) homogenizers, alas.  At this point, I am thinking that re-precipitating the samples with the 2-D cleanup kit is the way to go.  To prepare for this, I want to redissolve the jelly pellets (currently sitting in urea LB) with SDS.  Any suggestions what the best way to do this would be?  I was thinking of topping them up with an X% SDS solution (I don't mind increasing the sample volumes), how much SDS should I use?

ElkeKS

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Re: Pellet solubilization after TCA/acetone?
« Reply #11 on: July 22, 2011, 10:54:26 AM »
Hi Allison,

I had a look into Reiner's "Proteomics in Practice" book, where he lists SDS procedures under 'Alternative Lysis Solutions'. He says that up to 2% SDS has been used, and that the sample will have to be at least 20-fold diluted with urea and a non- or zwitter-ionic detergent before denaturing IEF. The SDS should then separate from the protein and migrate to the anode during IEF.
There are two references given:
Hughes GJ et al., Electrophoresis 13 (1992) 707-14
Görg A et al. in Simpson RJ Ed. 'Purifying proteins for proteomics: A laboratory manual' CSHL Press (2003) 407

Good luck!
Elke

Allison Mackay

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Re: Pellet solubilization after TCA/acetone?
« Reply #12 on: July 25, 2011, 08:35:11 AM »
Thanks, Elke.  I'll have to look both those books up, though I doubt we have them in the uni library here. 

I have replicated the problem with a test sample, it does seem to be related to the amount of protein precipitated at once.  So I'm going to try diluting those test samples and adding SDS to 2% and see if that helps them re-solubilize.  Then I'll 2-D Cleanup only a portion of that to use for IEF.

rachelnsw

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Re: Pellet solubilization after TCA/acetone?
« Reply #13 on: August 04, 2011, 12:43:15 AM »
This problem is the story of my (laboratory) life. I have plant samples that are ground in cooled PBS buffer with 5% b-mercaptoethanol. Then I do a spin at 20,800 x g for 5 mins at 4 C, take the supernatant and then acetone precipitate for 2h at -80 C or overnight at -20 C. THe pellet is impossible to deal with. I also see the jelly pellet. The chloro:methanol method also has an annoying pellet.

I will try 1 % pharmalyte and may alter my rehydration buffer so that I have 7M urea, 2M urea, 2.8mg dtt/mL, 2% CHAPS, 1% pharmalyte.

I'll let you know how it goes on a 1D gel. THanks everyone for your information.

FYI, this is my current method so you don't waste your time on it
I have tried rehydration buffer (6M urea, 2M thiourea, 2.8mg/mL DTT, 2% CHAPS) and 1 X sample buffer (2% SDS, 62.5 mM TrisHCl pH 6.8, 20% glycerol, 0.5 % bromophenol blue, 5 % b-mercaptoethanol) with eppendorf pestles, pippetting up and down and shaking on a rocker at 1400 rpm 25 C 30 mins. Then I do a spin 20,800 x g, 10 min, 4 C and take the supernatant for protein quantification. I get very small concentrations (0.5 ug/uL - 1.5 ug/uL) that are not good enough for my intended downstream applications.

Allison Mackay

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Re: Pellet solubilization after TCA/acetone?
« Reply #14 on: August 04, 2011, 01:13:06 PM »
Could adding Pharmalyte to the sample itself interfere with DIGE labeling later?  I assume not, but I should ask before I add it to my actual samples.  Also, I usually run in 0.5% Pharmalytes, which gives good focusing.  Would increasing this to 1% inhibit focusing at all?  Again, I assume not...  Or I could just reduce the Pharmalytes added to the rehydration buffer to compensate for that added to the samples.
« Last Edit: August 04, 2011, 01:16:15 PM by Allison Mackay »