Tutorial 7: Optimization.

Example with an additive screen

updated 6 September 2006

The first parameters to adjust are protein concentration, pH, and 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 l 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 l0x dilution of the additive.  Drops that stay clear can be seeded. 

 

Remember: you can click on any picture to get a bigger view.

 

 

Detergent 1:

n-octyl glucoside

 

 Rating: -

 Gave an amorphous, brown precipitate
 

 

 Detergent 2:

MEGA-8

 

Rating: -

Gave a horrible, black non-amorphous precipitate.
 

 

Detergent 3:

n-dodecyl glucoside

 

 Rating: 0

Gave a crystalline precipitate. Same as the control
 

 

Detergents 4 and 5:

Triton x-114

n-dodecyl-B-D-maltoside

 Rating: +

Both detergents gave thin crystals.

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