Preparing Tissue Culture Cell Extracts For Isoelectrofocusing

Preparation of samples for isoelectrofocusing containing relatively pure proteins is generally straightforward (see Basic Protocol 1, step 22). In contrast, complex samples such as whole-cell extracts, tissue extracts, or subcellular fractions are more difficult to prepare for successful isoelectrofocusing. Solubility limitations both prior to isoelectro-focusing and during focusing restrict analysis of these complex samples to protocols that include nonionic detergents and urea (see Basic Protocol 1). In addition, the presence of DNA and RNA in crude cell extracts further complicates isoelectrofocusing. The protocol presented below is suitable for preparing samples from cell cultures and is based on quantities compatible with silver staining or Coomassie blue staining. If smaller cell numbers and high-sensitivity detection methods such as autoradiography are used, volumes and quantities should be adjusted as needed.

In this protocol the cells are harvested and washed in phosphate-buffered saline (PBS) with proteolysis inhibitors, then lysed in Tris/SDS buffer using sonication, after which the total protein concentration is determined in the lysate. The lysate is further treated with a mixture of DNase and RNase, and additional SDS and reducing agent are added. At this stage, the samples can be stored at -80°C for an extended time or, after addition of urea and lysis buffer, may be loaded directly onto prefocused IEF gels. Filtration of the final sample prior to loading onto the IEF gel is essential for quality of isoelectro-focusing.

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