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Technical Tips for PhophoDetect™ Antibodies
 | | How do I increase my signal and decrease background staining? - To minimize background signal, cultivate the cells in serum-free or low-serum media for 24–48 hours before experimentation. In many cases, phosphorylation-dependent activation of signaling proteins can be detected only after serum starvation.
- Experimental protocols that do not include washing or centrifugation steps before cell lysis may improve results, because excessive manipulation of cells may lead to rapid activation of some signal transduction pathways.
- Use subconfluent cultures for induction of growth factor receptors to increase signal.
- Titrate the concentrations of primary and secondary antibodies to determine the optimal concentration.
- In general, do not use nonfat powdered milk or casein in blocking buffers or antibody diluent, because they contain numerous phospho-proteins that may lead to high background. Other blocking agents such as gelatin, BSA (Cat. No. 126593), ovalbumin (Cat. No. 32467), and serum (other than the species of primary antibody) may be used.
- Use cell lysis buffers that contain phosphatase inhibitors such as sodium orthovanadate (Cat. No. 567540) or sodium fluoride to maintain protein phosphorylation during cell lysis. PhosphoSafe™ Extraction Reagent (Cat. No. 71296) is specifically formulated to maintain protein phosphorylation during cell lysis.
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| | - Because phosphorylation is often a transient signaling event we strongly recommend
evaluation of the individual kinetics for each treatment. Peak signals may be obtained
any time from 2 minutes to 2 hours after induction, depending on the system being
studied.
| | | | - We recommend performing all steps between 15 and 22 °C. Incubation at lower temperatures
requires prolonged incubation time; higher temperatures will significantly
increase background staining.
| | | | - Using subcellular fractions for Western blotting, i.e., nuclei for nuclear proteins or
membranes for membrane receptors, may improve target protein detection. Try the
ProteoExtract® Subcellular Proteome Extraction Kit (Cat. No. 539790) to prepare four separate fractions from one sample: cytoskeletal, nuclear, membrane/organelle, and cytosolic.
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