| Conventional Western |
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Nano-Westerns - the CHIP approach |
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| 1. The blots were blocked for 1 hour in 5% skim milk powder in TBS containing tween-20. |
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1. The blots were blocked for 1 hour in 5% skim milk powder in TBS containing tween-20. |
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| 2. The primary antibodies (nonmuscle myosin, phosphothreonine and phosphoserine) were added to the blots in TBS-Tween, with 2% milk powder for 2 hours with gentle agitation. |
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2. The membrane was mounted in the CHIP and the same primary antibodies and concentrations that were used in the conventional Western were printed in an array, over the MHC. Each drop contained 100 pl, printed 0.15 mm apart, creating a line, 8 mm in length. |
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| 3. Excess primary antibody was removed by washing and then the HRP-linked secondary antibody was added for a further hour, followed by more extensive washing. |
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3. The CHIP was then used to wash the spots to remove non-specific binding and then a secondary antibody was printed in the same way as the primary antibody. |
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| 4. The interaction was detected using enhanced chemiluminescence reagents and film. |
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4. The interaction was detected using enhanced chemiluminescence reagents and film. |
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