There may be slight differences between the solutions in the screening kit and the ones you are using for optimization. Try to get the same lot numbers of solutions that were in the screening kit to use for the optimization.

Hi Sherif,

It depends on what is your goal.

If you would like to solve the structure, you need just one crystal.

If the crystal is too small and you would like to improve the quality, then

I would recommend to start from scratch. Forget about your crystals from kit.

Make your own solutions and control the purity of your samples and solutions.

Problem 1.

Let's imagine the best scenario: You got stock solutions identical to described in your kit from the same vendor.

The problem will be your water. You are going to dilute your stock solutions with water. And water is different even in the neighboring laboratory. And that could be the hardest thing to control.

Problem 2.

In many cases the pH listed in crystallization kit does not match real pH.

I had case when crystals grew from Sodium Potassium phosphate buffer. And it was very important how I prepared my buffer.

Problem 3.

In your case conditions look simple, but the problem could be very narrow range your protein crystalizes. pH could be 4.4 - 4.6, or PEG concentration 17.5 - 18.5 %.

If you built you screen kit with very small grid and did not get crystals I would think the problem would be a quality of water you are using.

George.

Dhiraj... sorry it is 100 mM

George Minasov

Thanks for your answer...

I will try to narrow down the range of pH and PEG concentration

about the water we use milliq water.. should I fitler it before use ?

Sherif,

If you can make your screen with reagents from the vendor which makes crystallization kit and avoid any additional additives (in your case water), you may get nucleation.

We had this case, when we got crystals from kit, tried to repeat conditions with their solutions, and no success. We gave up.

We found different solution, since we can not make the kit solution, we are using only kits. Our optimizations are variations of drop size, and ratio protein solution to crystallization solution.

One additional parameter you can screen would be amount of reservoir solution. The amount of reservoir solution effects on the volume of air in the crystallization compartment. More solution will make saturation faster. Processes in small drop go faster.

But to get big crystals you need bigger drops.

So, you have to try to play with:

size of drop

ratio

volume of reservoir

you can add temperature (if you can control that).

Good luck!

Hi Sherif,

1. Repeat the initial screening operation to eliminate the mutual contamination of the reservoir during the process of crystallization, especially in the screening of crystals using 96-well sitting drop crystal plate. At the time, due to improper operation or poor sealing, it is very likely that the reservoir will contaminate each other;

2. Please ensure the buffer, salt and precipitant used in the optimization are the same as those of the initial screening kit (including the conductivity of the ddH2O used to configure the buffer);

3. Try the method of hanging drops. The nucleation rate of hanging drop method is slower than that of sitting drop method, but the crystals formed are larger and more regular, which is more conducive to diffraction, which may be related to the volume and pressure change of droplets;

4. Inoculate the previously obtained crystals into droplets used to optimize the crystal to promote nucleation.

Good luck!