Protein aggregation and crystallization

From: anita p
Date: 26 August 2011 08:03

Hi All,
 I am working on a protein which has a membrane spanning region and a cytosolic domain. I have made various deletion constructs of the protein, so that I can have a crystallizable fragment.  There is no homologues mentioned in the pdb for this protein.
All of these constructs are purified successfully but when concentrated and loaded on a gel filtration column Superdex-200, they elute in the void volume.  But the proteins donot precipitate out.... !!
Is it worth while to go ahead for crystallization trials??
Any other suggestion is most welcome.
Thanks
Anita


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From: anita p

Hi,
I do have 10% glycerol in my buffers, and still the constructs come in the void volume.
and I have sarkosyl in the lysis buffer. but none in the elution or dialysis buffer. So do I still need detergents .... please suggest.
reg.
Anita

On Sat, Aug 27, 2011 at 12:13 AM, Pius Padayatti wrote:

Answer is simply no.
aggregates are of no value
why would you try it?
you could try adding glycerol to 10 % during the
preparation( all the way from homogenization) itself and try detergents
like c8e4 and series of that.
i know one membrane associated protein need
detergent from the begining itself.
Please do not chop off the membrane part keep it
and chop some of the unstructured cytosolic part if you want.
all the best.
let us know if any of these worked
Padayatti

--
Pius S Padayatti,PhD,

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From: anita p


Hi Yury,
I have done dynamic light scattering and it shows its polydispersed.
Please let me know if it is still ok for setting trays.
reg.
anita

On Fri, Aug 26, 2011 at 10:21 PM, Yuriy Patskovsky wrote:

Anita,
an assembly may be quite large - I would check it somehow, maybe by light scattering or centrifugation

Good luck

Yury

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From: Zheng Zhou


Hi, Anita

If you could find a way to test the elute's activity/binding to its' substrat/cofactor, then you will learn much more about your target. If the function assay is elusive, you could try superose column (5KDa-5MKDa). Does your light scattering tell you about the estimated size and MW?

Best,

Joe

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From: Allan Pang


Hi Anita,

Won't harm if you put it on crystallization tray. You never know what these proteins might do.

Allan

--
Allan Pang

PhD Student

G35 Joseph Priestley Building
Queen Mary University of London
London
E1 4NS