- Barbora set JCSG plate and got some 15 hit:
- To cluster them and understand if there are any trends in conditions that show crystalline precipitate (salt conditions vs PEG conditions vs alcohol conditions etc).
- Not to optimise with this protein batch.
- Next protein batch (20 x 20ul aliquotss @ ~13mg/ml) to be ready on Friday 03-11, then to do PCT test for 2,4,8,10,13 mg/ml and use that as reference for what crystallisation condition to use Barbora points out (wisely) that concentrating more could result in full protein crashout given concentrator requires 200ul starting material.
- Jose's note to self: how does the PCT translate to John William's suggestion to get crashout vs OD scheme.
Monday, 30 October 2017
20171030 weekly catch up
20171030
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Summary of chat with Jo N with reminders about crystallisation:
ReplyDelete* PCT informs whether there's hope to crystallise and wheter prot is too dilute
* Initial coarse screen should deliver mix between clear and precipitate
** One can learn trends from both the conditions that DID deliver xtals as well as the ones that DID NOT deliver crystals, like: protein did/not like salt, like/not high pH etc
Barbora tried a JCSG screen at around 30-Oct. She got a mix of clear and precipitated drops (good lead).
ReplyDelete* Jose suggested to survey the tray through Xtal Pims and highlight trends of which conditions gave good crystalline-looking material.
* Afterthought: check trends of which conditions resulted in clear drops as well.
* Barbora to repeat screen with new prep as JCSG block used was mouldy and old batch protein is finishing.
* New batch: PCT 2,4,6,8,10,13% and fresh JCSG. Then survey and compare hits to the mouldy JCSG and optimise around best looking ones.
purification Tuesday 7th, then cleavage and second purification. In the end of the week should be able to conduct PCT test and maybe readyfor xtalisation.
ReplyDelete