In-vitro Ubiquitination [View Printable]
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gsovak
Group: Member
Posts: 544
Joined: Jan 25, 2005
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Hi to all,
Is anyone doing/done an in-vitro ubiquitination assay?
I would like to start to do this assay and would like to hear from people who did it before.
Thank you
Guy |
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| Posted Dec 20, 2006, 23:04 PM |
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samm
Group: Moderators
Posts: 407
Joined: Mar 03, 2005
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Hi Guy! BioMol/Affiniti research have a ubiquitinylation and many de-ubiq. / enzyme assay kits - you might want to check out their web site. I don't know exactly what you want to do, but check out Cat#UW9920 |
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| Posted Dec 30, 2006, 2:06 AM |
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gsovak
Group: Member
Posts: 544
Joined: Jan 25, 2005
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Hi Samm,
I want to start doing in vitro Ubiquitination assay, and to check the importance of Heat shock chaperones by adding Reticulocyte lysate.
Did you ever done some in vitro Ubiquitination?
Thanks
Guy |
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| Posted Jan 01, 2007, 16:38 PM |
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gsovak
Group: Member
Posts: 544
Joined: Jan 25, 2005
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Hi again Samm,
I looked into the product and it seems that it would help me.
Did you ever used it, Do you have some tips.
I am going to use BHK lysate.
Thank you for all tha information
Guy |
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| Posted Jan 01, 2007, 16:45 PM |
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samm
Group: Moderators
Posts: 407
Joined: Mar 03, 2005
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Hi Guy! I've never used it personally, and the only person i know who's done it is still away (and as far as I remember it was a non-kit thing - so the kit might make things easier). Can you check with the company - the one Ab I've used was excellent. |
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| Posted Jan 02, 2007, 20:57 PM |
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biocat
Group: Member
Posts: 1
Joined: May 19, 2007
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hi there, I have done in vitro , what are your questions? |
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| Posted May 19, 2007, 14:29 PM |
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gsovak
Group: Member
Posts: 544
Joined: Jan 25, 2005
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Hi ,
Did you work with RRL and did some ubiquitinaation? |
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| Posted May 20, 2007, 5:11 AM |
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cs
Group: Member
Posts: 2
Joined: Feb 02, 2008
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| biocat said: | | hi there, I have done in vitro , what are your questions? |
Hi I am doing InVitro Ubiquitination assay because my major is phytochorme biology...phytochromeA is ubiquitinated by one of the repressor protein COP1..this COP1 working as E3 Ligase..so basically we are using purified COP1 as E3 ligase and adding our purified PhytochromeA...we are initiating the experiment with adding 1 ug of Ubiquitin for 40 ul total reaction...we are adding E1 1.6 ul (from 25ug -UBE1 rabbit-Boston biochem) and E2 0.8 ul (from 50 ug UBcH5b-Boston biochem) and E3 (our COP1) 1 to 2 ug..100 mM Tris-cl pH7.4..5-10 mM ATP 5-10 mM MgCl2..0.5 to 1 mM DTT..and our COP1 need ZnCl2 (because this COP1 contain zinc finger motif) so 20 to 50 uM ZnCl2..this is reaction condition.. but we are not getting any polyubiquitination bands with PhyA ...we are using some times GST Ubi also for western detection.. please give some suggestion..because COP1 act as E3 ligase..we need to change any E1 or E2 concentrations or energy or we are not getting key point... please clarify...thank you in advance.. |
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| Posted Feb 03, 2008, 4:48 AM |
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