1. How much protein do I need to run the OptiSol solubility screen?

2. Which Protein assay can I use?

3. Can I process a membrane protein sample with OptiSol?

4. Will my aggregated protein sample 'go in solution' with the OptiSol reagents?

5. My protein sample 'crashes out of solution' when I leave it on ice over night - can OptiSol help?

6. I need more information. Send me your product literature.

1. How much protein do I need to run the OptiSol solubility screen?

This is a crucial factor to successfully completing the OptiSol Protein Solubilization Screen. Fundamentally, the protein concentration to use depends on the sensitivity of the protein assay that will be employed. Protein solutions with concentrations of >10 mg/ml or <1 ug/ml are fine, as long as the assay detection limit is compatible.

Back-of-the-envelope calculation to estimate the protein concentration that is necessary for a particular protein assay.

The OptiSol protocols call for dilution of the protein sample in 96 wells. Dilution is ca. 1:15, using the supplied OptiSol formulations. Let's say you've got a 1 mg/ml protein solution and for each well in the 96 well plate you'll dilute 10 uL of your protein solution with ca. 150 uL of formulation. You'll end up with a diluted protein solution of ca. 0.07 mg/ml.

This is the maximal concentration that one would obtain for 100% solubilization. Let's say your solubilization yield is less, let's say 10%. That's 7 ug/ml. The purpose of the OptiSol screen is to rank 90 different formulation for their solubilization efficiency. This requires to differentiate between protein contents in different wells. Therefore an assay that can reliably detect less than 1 ug/ml of protein would work well.
To make a long story short, we advise to employ a protein concentration that one can comfortably detect when the concentration is lowered by ca. three orders of magnitude

2. Which Protein assay can I use?

Any protein assay that has sufficient specificity, dynamic range and sensitivity. Some researchers run dot-blots (Western), some have a fluorescence readout, employ an ELISA or use an enzymatic activity assay. This is up to you and depends on what you want to screen for.
Functional assays (measuring binding or enzymatic activity) or passive assays (measuring protein mass) can both be employed.

3. Can I process a membrane protein sample with OptiSol?

OptiDet is better suited to determine the optimal detergent formulation to keep a membrane protein solubilized, while minimizing aggregates.

4. Will my aggregated protein sample 'go in solution' with the OptiSol reagents?

This depends on the nature of the protein aggregate. The only way to find out is by experimental testing - and this is exactly what the OptiSol protein solubility screen does. Test 90 different mild formulations - different pH, salts, polymers, sugars, polyols, detergent, reducing agents. If the protein sample is reversibly aggregated, chances are the OptiSol screen will yield conditions that solubilize the sample.

The formulations employed are rather mild. The goal is to solubilize the aggregate and yield functional protein. Will any aggregated protein samples get solubilized? No, some aggregates just contain 'dead' protein. You'll need SDS or urea for such recalcitrant samples, and obtain non-functional protein in return.

5. My protein sample 'crashes out of solution' when I leave it on ice over night - can OptiSol help?

YES! The buffer you currently use is not optimal since it leads to protein aggregation. How to identify which ingredients protect the protein from 'crashing out'? By systematically testing the precipitation behavior in 90 different mild formulations - different pH, salts, polymers, sugars, polyols, detergent, reducing agents. Chances are this OptiSol screen will yield conditions that minimizes sample aggregation.

What else would you like to know?

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