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During preparation of ß-Lactoglobulin solution, the following points should be noted:
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The ß-Lactoglobulin Control is supplied as a lyophilized powder (16 mg). Immediately before use, reconstitute the control in 500 µl of 1X Assay Buffer to generate a 32 mg/ml stock solution.
- Gently mix to re-suspend the ß-Lactoglobulin in solution. Mix the solution in a rotator for about 20 minutes to
completely resuspend the protein. It is preferable to centrifuge at maximum speed for about 30 seconds to allow undissolved materials to separate and collect at the bottom of the tube. - Do not vortex and avoid formation of bubbles. It is recommended to perform the assay as soon as possible after re-suspending the ß-Lactoglobulin Control.
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Recommended storage for unused solution of ß-Lactoglobulin is 4°C for one month. Freezing should be avoided since this could cause some denaturation.
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If storage of ß-Lactoglobulin solution for longer periods is necessary, it should be aliquoted in smaller volumes and stored at -20°C. Once thawed, each aliquot should be used and the remaining unused solution should be discarded.
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The final concentration of the ß-Lactoglobulin solution for use in the assay should be 16 mg/ml. For most assays, the protein concentration can be within the range of 1-16 mg/ml, depending upon the protein being used. Ideally, the concentration should be closer to 1 mg/ml. Note that aggregates above 20 mg/ml can saturate the dye.
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Note that additives such as glycerol, different buffer compositions and different protein concentrations can affect the aggregation temperature.