cOmplete™ ULTRA Tablets, Mini, EDTA-free, EASYpack Protocol & Troubleshooting

Product No. 05892791001

Protocol

Removal

All inhibitors can be removed via dialysis. Use a membrane with cutoff >10,000.

Heating to +60 °C for 60 min has not been tested. However, it is expected that the activity will be decreased considerably. Add one tablet again after this heating step.

Protein Determination

The BCA-Test from Pierce is recommended for determining the protein concentration. Please be aware that the absorbance value may be increased (approximately 100 mE higher absorbance). Therefore, the determination of a standard curve in the specific media (for example, with BSA) is recommended. Nevertheless, the linearity of the standard curve (absorbance vs. concentration) should not be influenced negatively.

Western Blot

Detection of protein via western blot were evaluated during the development. The pattern and the intensity of the detected bands were identical.

Carefully push the tablet through the foil packaging using the base of your thumb (not your fingernail) to prevent the breakage of tablets.

Prepare a stock solution (2x) or add the tablet directly to the buffer.

Troubleshooting

Inhibition does not work

Increase of the concentration of the inhibitor cocktail and reduce the storage time.

Also, note that some ′′rare′′ proteases might not be inactivated. In general, it cannot be guaranteed that all proteases will be blocked. In some special cases, the addition of specific inhibitors may be indicated. In those cases, the customer must screen for the right inhibitor.

Protein not found in western blot

The loss of protein may be due to other factors:- Subunits that are separated (for example, as a result of using reduced conditions).- Inaccurate blotting conditions.- Detection problems.

Additional bands in western blots

Try running a blank on the gel containing only the inhibitor cocktail.If the inhibitor cocktail has been used at a very high concentration (other than as advised in the package insert), there may be slight bands in the low molecular weight region.

Interference with isoelectric focusing

This might be caused by one of the components that may be visible on the gel after staining. Apart from that, nothing else should disturb this application; try using a blank to verify this.

Inhibition of inhibition activity

Theoretically, there is no reason to believe that SDS or other detergents are not compatible with the inhibitor cocktail. However, no direct experiments have been carried out on this subject.

Note that Ca2+, Co2+ Ni2+, Mn2+, and Mg2+ can inhibit the inhibition activity.

Recommended solvent

One tablet can be dissolved in 5 ml double-distilled water. If the tablet will be dissolved in lysis buffer extra time may be required. Leaving the solution at +15 to +25 °C for 30 - 60 minutes or vortexing may help.

Interference with DTT

This has not been checked. From the components of the tablet, we would expect that reducing agents normally used in protein biochemistry (like mercaptoethanol, DTT up to 5 mM, or DTE) should not interfere with the function of the tablets.

Materials

     

COMPLETE is a trademark of Roche

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