Western Blot - Cell Lysate Protocol

This protocol is intended to provide a set of initial conditions for analysis of cell lysates samples by Western blot. Further optimization may be required for individual samples or analytes. Follow manufacturer's protocols for specific reagents when applicable.

Preparation of Cell Lysates for Western blots:

Prepare total cell lysates by solubilizing cells in an appropriate sample buffer, such as 2X SDS sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, 10 mM NaF and bromophenyl blue), at approximately 2x10⁶-1x10⁷ cells per mL. The extracts are heated in a boiling water bath for 5 minutes and then sonicated with 3-4 bursts of 5-10 seconds each.

Primary Antibody Selection & Optimization

The most important factor when designing an IHC/ICC experiment is selection of the primary antibody. In turn, the critical feature of a primary antibody is specificity for the epitope. All steps of an IHC/ICC experiment must be optimized to visualize specific staining and minimize non-specific background signals. This includes performing initial studies to determine the appropriate incubation conditions for each primary antibody. The working dilution for an antigen affinity-purified polyclonal antibody is generally lower than that of a monoclonal antibody but these values must be determined empirically. To achieve a robust and specific signal, a high quality antibody that exhibits minimal cross-reactivity should be employed.

Starting conditions for Primary Antibody Incubation

	Monoclonal Antibody	Polyclonal Antibody
Tissue	5-25 µg/mL, overnight at 4 °C	1.7-15 µg/mL, overnight at 4 °C
Cells	5-25 µg/mL, 1 hour at room temperature	1.7-15 µg/mL, 1 hour at room temperature
Advantage	Single epitope specificity	Lower concentration required
Limitation	Vulnerable to epitope masking	Heterogeneous population

Protocol for Making a 4% Formaldehyde

The vast majority of IHC/ICC procedures employ fixation of tissues and cells using formaldehyde-based fixatives. The protocol below describes the technique for generating a 4% formaldehyde solution in PBS. The most effective fixative must be determined experimentally.
Caution: Formaldehyde is toxic. Please read the MSDS before working with this chemical. Gloves and safety glasses should be worn and solutions made inside a fume hood.

Please read the protocol in its entirety before starting.

Protocol for the Preparation & Fixation of Cells on Coverslips

Source : www.rndsystems.com