- Dilute protein samples in buffer to final protein concentrations of 1–100 ng/µl.
Tip: The protein of interest is diluted in dilution buffer for denaturing conditions, dilution buffer for native conditions, or another preferred buffer.
- Apply 1 µl samples of diluted protein directly onto membrane. It is also possible to use crude cell lysate and apply 1 µl samples with an estimated concentration of 1–100 ng/µl protein.
Note: Under native conditions especially, the antibody epitope must be at least partially exposed to allow antibody binding. In most cases diluting the protein with buffer containing denaturing reagents will increase epitope exposure and give better results.
Tip: To differentiate between nonspecific and positive signals, an extra sample containing 1 µl of a cell extract of the host strain without plasmid (or other suitable control) should also be applied to the membrane and treated together with the protein of interest.
- After applying the samples, the membrane should be dried for a short time at room temperature before proceeding with the detection process.
Tip: For larger sample volumes, suitable equipment is available from several suppliers.
- Proceed with immunodetection (see Immunodetection using a chemiluminescent detection method or Immunodetection using a chromogenic detection method).