Cell Lysis and Fractionation Support—Troubleshooting

Consider the sample type: some cells are easier to lyse than others. In animal cells, the plasma membrane is the only barrier separating cell contents from the environment, but in plants and bacteria the plasma membrane is surrounded by a rigid cell wall. Animal cells therefore lyse fairly readily, whereas plant cells are particularly strong and difficult to lyse. The technique chosen for disruption of cells must take into consideration the origin of the cells or tissues being examined. Physical disruption may be necessary for efficient lysis of certain cell types.

Detergent removal can be attempted in a number ways. Dialysis is effective for removal of detergents that have very high CMCs (critical micelle concentrations) and/or small aggregation numbers. Detergents with low CMCs and large aggregation numbers cannot be dialyzed since most of the detergent molecules will be in micelles that are too large to diffuse through the pores of the dialysis membrane; only excess monomer can be dialyzed. Ion exchange chromatography using appropriate conditions to selectively bind and elute the proteins of interest is another effective way to remove detergent. Sucrose density gradient separation also can be used. See our Tech Tip for removal of detergent from protein samples.

Try using a protease inhibitor and perform all lysis steps at 4˚C.

If the protein is being transfected into the cell line, you may need to further optimize the transfection procedure. You should also be sure you are using the recommended amount of lysis reagents. Finally, increasing the incubation time or more vigorously mixing the sample during incubation can aid in cell lysis.

See details of the various B-PER™ reagents we offer.

Some overexpressed proteins can be insoluble, misfolded, or expressed in inclusion bodies. The best way to obtain soluble protein is to adjust the expression conditions; however, if this is not possible, the Inclusion Body Solubilization Reagent (Cat. No. 78115) can be used.

We recommend using B-PER™ Complete Bacterial Protein Extraction Reagent, an all-in-one formulation that combines a lysis reagent with lysozyme and a universal nuclease to enable mild extraction from both gram-negative and gram- positive bacteria. If the addition of lysozyme and DNase I might interfere with your downstream application, we recommend using other B-PER™ reagents. See details of the various B-PER™ reagents we offer.

The temperature may be too low; warm the culture and other reagents to room temperature. The lysozyme may have low activity or be inactive. Add more lysozyme to the reagent or purchase new enzyme.

Add DNase I to the B-PER™ solution. If DNase was added, the enzyme may have low activity or be inactive. You can add Mg2+ ions to 2 mM final concentration, add more DNase I to the reagent, or purchase new enzyme. Alternatively, use B- PER™ Complete reagent, an all-in-one formulation that combines a lysis reagent with lysozyme and a universal nuclease to enable mild extraction from both gram-negative and gram-positive bacteria.

Many proteins spontaneously refold into their native, functional structures when the denaturing solubilization reagents are removed by dialysis. Other proteins, however, will fold into non-functional and even insoluble forms by this process. In such cases, specialized sets of buffer conditions must be tested to identify those that promote the highest possible yield of properly refolded protein. See our Pierce™ Protein Refolding Kit (Cat. No. 89867).

Some overexpressed proteins can be insoluble, misfolded, or expressed in inclusion bodies. The best way to obtain soluble protein is to adjust the expression conditions; however, if this is not possible, the Inclusion Body Solubilization Reagent (Cat. No. 78115) can be used.