microseeding

From: Rajesh kumar
Date: 19 March 2012 20:31

Dear All,

I have few papers in hand which  explain me about microseeding, matrix microseeding, and cross seeding.

I have also read few earlier threads and some more literature in google.

Using Phoenix robot, I did a matrix micro-seeding and matrix cross seeding. I have few hits with this.

In 96 well I used 100+100+50 nL and 200+200+50 nl (protein+screen+seed) in separate expts.

I have hard time to plan to translate this 96 sitting drop well plate to 24 well plate to refine the conditions to get better crystals. only 1-2 hits are small crystals and they are tiny. 

 I wonder in 24 well plate, if I should do-

1)  for Example 500+500+50nl (I am sure I cant add less that 500 nL precisely)

2) to a drop of 500+500 nL do microseeding/streaking with a hair

I appreciate if you could advise and share some practical ways to further my experiment.

Thanks in advance

Regards,

Rajesh

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From: Ed Pozharski


Scaling up 100nl drops is problematic.  What I understand is that it is
not only the different equilibration conditions, but primarily the
amount of protein that gets absorbed on the surface is relatively higher
for small drops.  There were some empirical formula for scaling up (i.e.
how much you need to increase the protein concentration, but I am afraid
you would have to re-screen anyway.

--
Edwin Pozharski, 

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From: Patrick Shaw Stewart



Rajesh

If you set up the volumes you suggest you will probably get precipitation.  This is counterintuitive until you realize that (as Ed says) you will be losing a lot of protein with those small drops.  When you scale up the surface area to volume ratio is lower, so a smaller proportion of the protein is lost.  Therefore you go up on the phase diagram and get precipitant or very small crystals.

Normally halving the amount of protein for the hits from 200 nl drops works (suggesting that half the protein is lost from such small drops).  Try say 500+1000+500 (don't reduce the volume of seed stock because the solution that you suspended the crystals in may be important).  Or dilute the protein and use 1000+1000+500.

For the hits from the 450 nl drops you could reduce or dilute the protein by say 25.%.

Or make plenty of seed-stock and try seeding into a random screen again with larger drops, say 1.5+1+0.5 ul

Those tiny crystals should be good for seeding, don't worry about that (provided they are protein of course).

Streak seeding may work but bear in mind that roughly a third of the precipitant comes from the seed stock in your 250 nl drops.

You can add the seed stock with a syringe and needle if you don't have suitable robot ;)

Experience and data-mining suggests that reducing the salt precipitant (in high-salt drops) or salt additive (in PEG drops) by around 50% may be helpful too when scaling up - I'm not sure why this works.

Good luck

Patrick








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From: Patrick Shaw Stewart



Sorry, I said that last part wrong.  I meant it is sometimes helpful to increase the salt by 50% when scaling up.

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From: Enrico Stura


Patrick,

This thing all things devours:
 Birds, beasts, trees, flowers;
 Gnaws iron, bites steel;
 Grinds hard stones to meal;
 Slays king, ruins town
 And beats high mountains down.

 Answer: TIME

When scaling up in seeding  the most important factor is: "TIME"
While the environment may be the same, the rate at which it changes
is not. Since crystal growth takes TIME, the change in volume will
affect supersaturation in a time-dependent manner.

Using salt or any other parameter to compensate for TIME will
introduce an extra variable which you may not know what its effect
will be.

Sorry to mess up your solution.

Enrico.