native protein extraction protocol

1. Native protein extraction protocol Native protein isolation are required for various experimental applications such as in vitro biochemical assays and structural studies. Both the quantity and purity of native protein have to be sufficient for downstream analysis. Successful native protein isolation is able to preserve the native activity and three-dimensional structure of a target protein during harvest. Chromatography is the major technology for native protein isolation. Four basic steps of native protein isolation cell lysis; protein binding to a matrix; washing; elution. Common techniques for native protein isolation Affinity Chromatography Ion Exchange Chromatography Gel Filtration Chromatography Reverse-phase chromatography Troubleshooting: Problem After elution, there is no recombinant protein recovery. The loading amount of sample is not enough. Increase the amount of sample loaded or lysate used Protein degraded. Perform all the native protein isolation steps at 4°C Make sure that the His-tag is not cleaved during native protein isolation Add protease inhibitors during cell/tissue lysis. The affinity between recombinant protein and resin is too high. Increase stringency of elution by decreasing the pH Increase the imidazole concentration Use EDTA or EGTA (10– 100 mM) to strip resin of nickel ions and elute the protein in order to preserve activity Native protein was washed out by too stringent washing. extensively with wash buffer at an increased pH Expression level is too low. Optimize your expression protocols follows the guidelines There is no protein bonded because of the protein folding. Try denaturing conditions Good recombinant- protein recovery but with non-recombinant proteins contamination. Wash conditions are not sufficiently stringent. Cause Solution Wash less Wash more

2. extensively with wash buffer at a decreased pH Recombinant protein has low affinity for resin; comes off in wash with many contaminating proteins.Try denaturing conditions Perform an imidazole gradient elution Perform a decreasing pH gradient Sample contain other His-rich proteins. Before perform elution, try an additional high stringency wash at a lower pH (for example, between pH 4 and pH 6) Further purification of the eluate on a new column after protein dialysis of the eluate against the binding buffer and proper equilibrate the column with binding buffer Use another type of column to perform the native protein isolation one more time Native protein was washed out by too stringent washing. is too low. Before perform elution, try an additional high stringency wash at a lower pH (for example, between pH 4 and pH 6) Recombinant protein do not bind to resin tightly. Try reverse-chromatography which only works for native protein isolation Column become white. Buffers with Chelating agents which strip the nickel ions from the column. The column need to recharge thoroughly Column become reddish brown. Buffers with DTT. reducing agent Detect recombinant protein in the flow through and wash fractions. Protein overload. Reduce the amount of protein loaded on the column or increase the amount of resin for native protein isolation Protein precipitation occurs during binding. Temperature is too low. Perform native protein isolation at room temperature. Protein aggregation occurs.You may add solubilization reagents such as 0.1% Tween-20 or 0.1% Triton X-100 or stabilizers such as Mg2+. All buffers may need to add solubilization reagents to maintain protein solubility. Run column in drip mode in order to prevent protein from dropping out of solution. Besides native protein isolation, Creative Biostructure is also pleased to assist you in all process of protein purification project with other corresponding technical resources. Please see the table below for more details and feel free to Contact Us. Expression levels Try denaturing conditions Use β-ME as a Related Sections Services: Plasmids Design and Construction Protein Production and Purification Protein Engineering Service

3. Protein Analysis Native Protein Purification Protein Process Development and Manufacturing Protein Crystallization and Structure Determination Custom MemPro™ Membrane Protein Services