Troubleshooting of Cleavage Methods
The troubleshooting guide below addresses problems common to the majority of cleavage methods as well as problems specific to a particular method. In the latter case, the relevant method is indicated.
Problem | Possible cause | Solution |
---|---|---|
GST-tagged proteins are not cleaved completely. | The ratios of PreScission Protease, thrombin, or Factor Xa to GST-tagged protein are not optimal. | For PreScission Protease and thrombin, use at least 10 units/mg of tagged protein. For Factor Xa, use an amount equivalent to at least 1% (w/w) of the weight of tagged protein. For some tagged proteins, up to 5% Factor Xa can be used. The optimal amount must be determined empirically. In some cases, optimal results have been achieved with a tagged protein concentration of 1 mg/mL. The addition of ~0.5% SDS (w/v) to the reaction buffer can significantly improve Factor Xa cleavage with some tagged proteins. Various concentrations of SDS should be tested to determine the optimal concentration. |
The incubation time is not sufficient for complete cleavage of the protein from the GST tag. | Increase the incubation time for the cleavage reaction. Increasing the reaction time to 20 h or more should improve cleavage as long as the tagged protein is not degraded by the extended incubation period. | |
Specific cleavage sites for the proteases have been altered during cloning of the tagged protein. | Verify the presence of specific enzyme cleavage sites. Check the DNA sequence of the construct and compare it with a known sequence to verify that the cleavage sites have not been altered. | |
The presence of cleavage enzyme inhibitors is interfering with the cleavage reaction. | Remove any enzyme inhibitors that may interfere with the cleavage reaction. Prior to cleavage with PreScission Protease, buffer exchange or dialyze the tagged protein against 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.5. Prior to cleavage with Factor Xa, buffer exchange the tagged protein on a desalting column, or dialyze against 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5. | |
Factor Xa is not properly activated. | Factor Xa protease is preactivated. If using a protease from another source, activate Factor Xa protease with Russell´s viper venom to generate functional enzyme. For activation of Factor Xa protease, incubate Russell´s viper venom with Factor Xa protease at a ratio of 1% in 8 mM Tris-HCl, 70 mM NaCl, 8 mM CaCl2, pH 8.0. Incubate at 37 °C for 5 min. | |
The first amino acid after the Factor Xa protease recognition sequence is Arg or Pro. | Check the sequence of the tagged protein to verify that the first three nucleotides after the Factor Xa protease recognition sequence do not code for Arg or Pro. | |
Multiple bands are observed after electrophoresis/ Western blotting analysis of the cleaved target protein | Proteolysis is occurring in the host bacteria prior to the cleavage reaction. | Determine when the extra bands appear. Verify that additional bands are not present prior to PreScission Protease, thrombin, or Factor Xa protease cleavage. |
The tagged protein itself contains recognition sequences for PreScission Protease, thrombin protease, or Factor Xa protease. | Check the sequence of the tagged protein to determine if it contains recognition sequences for the cleavage enzymes. | |
The tagged partner is contaminated with protease after purification | Glutathione Sepharose may have been saturated with GST-tagged protein during purification. | Pass the sample over a new GSTrap column or fresh Glutathione Sepharose to remove residual PreScission Protease, or over a HiTrap Benzamidine (high sub) column in the case of thrombin protease or Factor Xa protease. |
Materials
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