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PKA-alpha-Western-blot - Revision history
2026-10-18T08:04:47Z
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Paul.xu at 12:41, 25 May 2011
2011-05-25T12:41:26Z
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<p><b>New page</b></p><div>1. SDS-PAGE<br> <br />
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Make sure to load 5 ul of color protein marker<br> <br />
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<br> <br />
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2. Transfer<br> <br />
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use transfer buffer with 10% methanol<br> <br />
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25 v for 1 hour<br> <br />
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<br> <br />
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3. Blocking<br> <br />
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TBST with some milk powder for 15 min, then rinse with water and TBST<br> <br />
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<br> <br />
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4. 1st antibody<br> <br />
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Rabbit anti-PKA, 1:2000, add 1 ul to 2 ml TBST, incubate (shaking gently) at RT for 1 h.<br> <br />
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<br> <br />
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5. Wash<br> <br />
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rinse once with water, then 10 ml TBST, 3 times, 5 min each<br> <br />
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<br> <br />
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6. 2nd antibody<br> <br />
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AP conjugated Goat anti-Rb, 1:10000, (0.5 ul to 5 ml TBST)<br> <br />
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RT for 30 min gentle shaking.<br> <br />
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<br> <br />
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7. Wash<br> <br />
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rinse once with water, then 3 times of 10~15 ml TBST, 5 min each.<br> <br />
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<br> <br />
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8. Develop<br> <br />
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Rinse with water, add 5 ml of AP buffer, 50 ul BCIP/NBT Color Development Substrate.<br> <br />
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stop reaction until you see brown bands. <br />
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<br> <br />
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'''Alkaline Phosphatase Buffer (AP Buffer)'''<br> <br />
<br />
100mM Tris-HCl [pH 9.0], 150mM NaCl, 1mM MgCl2)<br> <br />
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'''NBT/BCIP''' <br />
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18.75 mg/ml NBT (Nitro blue tetrazolium chloride) and 9.4 mg/ml BCIP (5-Bromo-4-chloro-3-indolyl phosphate toluidine salt) in 67% DMSO <br />
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[[Category:Protocols]]</div>
Paul.xu