Optimization

Tutorial 7

Example with an additive screen

The first parameters to adjust are protein concentration, pH, an precipitant concentration. Once you have the mother liquor cocktail (buffer, precipitant) and the protein concentration more or less well defined, you can screen additives.

Step 1: Fill a 24-well plate with 24 x 1 ml of the optimized mother liquor cocktail in each reservoir. This insures that the reservoirs are identical as possible and that what you will now be testing are the effects of the additives and not your ability to pipette accurately.

Step 2: Take one of the excellent detergent or additive kits from a commercial vendor or make your own. In the pictorial example here I was screening the effects of different detergents. Therefore, each drop contained:

2.5 ul protein + 2.5 ul reservoir (working solution) + 0.5 ul detergent

I set up 23 drops with detergent and l drop without (as a control.)

Step 3: After an appropriate time, examine the drops. I use a simple, 4-scale rating system.

+

means it is better than the control

0

means the drop stayed clear

=

means it gave the same result as the control drop

means it is worse than the control

If you don’t get any plusses, then work with the minuses. At least it means the additive is affecting the system. Try repeating with a ten-fold dilution of the additive. Drops that stay clear can be seeded.

Here are four examples using the same conditions but different detergents:

Tutorial 7

Example with an additive screen

The first parameters to adjust are protein concentration, pH, an precipitant concentration. Once you have the mother liquor cocktail (buffer, precipitant) and the protein concentration more or less well defined, you can screen additives.

Step 1: Fill a 24-well plate with 24 x 1 ml of the optimized mother liquor cocktail in each reservoir. This insures that the reservoirs are identical as possible and that what you will now be testing are the effects of the additives and not your ability to pipette accurately.

Step 2: Take one of the excellent detergent or additive kits from a commercial vendor or make your own. In the pictorial example here I was screening the effects of different detergents. Therefore, each drop contained:

2.5 ul protein + 2.5 ul reservoir (working solution) + 0.5 ul detergent

I set up 23 drops with detergent and l drop without (as a control.)

Step 3: After an appropriate time, examine the drops. I use a simple, 4-scale rating system.

+

means it is better than the control

0

means the drop stayed clear

=

means it gave the same result as the control drop

means it is worse than the control

If you don’t get any plusses, then work with the minuses. At least it means the additive is affecting the system. Try repeating with a ten-fold dilution of the additive. Drops that stay clear can be seeded.

Here are four examples using the same conditions but different detergents:

n-octyl glucoside

MEGA-8

n-dodecyl glucoside

Triton x-114 and n-dodecyl-B-D-maltoside