Protein Protocols Protein Protocols

Western Blotting Using ECL 435
Table 1
Calculation of Unknown Actin Concentrations Using Data from Images Generated
by Storm and ImageMaster VDS-CL. For images created on film and analyzed
by the PDSI the unknown was outside the linear range of the standard curve
Volume rfu Calculated ng Actual ng
Storm:
Sample
Actin unknown 938162 61 54
CCD camera:
Sample
Actin unknown 3582848 56 54
2. As the optimum exposure time may not be known, it is recommended to take several
exposures of increasing time in rapid succession. This avoids losing signal while appropriate
exposure times are estimated.
3. Quantification can then be performed by exporting the image as a 12–16-bit file into a
suitable image analysis program to measure band intensities.
4. Notes
4.1. Preparation of Protein Blots
1. For accurate quantification a dilution series of protein standards is required. This is easily
done if the purified protein is commercially available; otherwise cloning and expression
may be required.
2. If using film to image the Western blots, the dilution series should extend over a 10–20-fold
range. For detection using fluorescence scanning or a CCD camera, a dilution range of up
to 50-fold is recommended owing to the increased linear dynamic range achievable.
3. For greater accuracy it is recommended that duplicate gels be used for analysis. The running
of several concentrations of the test sample will also help to increase the accuracy of
quantification.
4. Overnight electroblotting is generally more reproducible than shorter blotting at higher
voltages. However, the optimum procedure will depend on the particular protein being
analyzed. Blots may be used immediately or air-dried and stored in a desiccator in a
refrigerator (2–8°C) for up to 3 mo.
4.2. Immunodetection
5. PVDF membranes will require rewetting in methanol if they have been allowed to dry out
after electroblotting.
6. The use of 5% nonfat dried milk in PBST is suitable for most applications but, for certain
antigen antibody systems, alternative blocking conditions may need to be used.
7. The dilution of the primary antibody required to give optimum results will vary and should
be determined for each antibody used. These optimization experiments may be performed
by dot blot analysis.
8. Where backgrounds are still high, the inclusion of 0.25–0.5% blocking reagent in the
antibody incubation may sometimes improve backgrounds.
9. As a general rule, as large a volume as possible of washing buffer should be used each time.
10. Due to the sensitivity of the detection system, the dilution of the HRP-labeled second
antibody should be optimized to give the highest signal with minimum background. A