PKC western blot - (Mar/01/2006 )
I'm wanting to perform a western blot for PKC-alpha. Does anyone have a protocol or any suggestions as to special conditions and what antibodies to use?
Here is my protocol, I use Santa Cruz PKC alpha rabbit antibody and the anti rabbit IgG although I know some people have a problem with them:
Gel Electrophoresis of Protein Samples
4% polyacrylamide stacking gels and 12% separating gels were used to perform Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) based on a method described in 1970 by Laemmli. The following protocol was used:
Gels were cast in a glass mould (see table 1 for gel make up) initially 10ng of protein sample (determined by Lowry microassay) was denatured by boiling in 5μg of SDS loading buffer (125μM Tris, pH 6.8, 25% Glycerol, 7.5% β-mercaptoethanol (added just before addition to protein), 5% SDS, 0.5% bromophenol blue) for 7mins. The samples were loaded onto the gel with a protein marker in the first lane and the gels run in a running buffer (250mM glycine, 25mM Tris, 0.1% SDS (pH 8.3)) at around 200 volts. Samples were run for 50mins.
Table1. Make up of polyacrylamide gels (4 gels)
Gel size 7% 9% 12% 15%
Acyle:Bis (29:1) 3.3ml 4.4ml 5.7ml 7.1ml
Tris 1.5M pH 8.8 4.6ml 4.6ml 4.6ml 4.6ml
Distilled H2O 10.9ml 9.7ml 8.4ml 7.0ml
APS 10% 150μl 150μl 150μl 150μl
TEMED 15μl 15μl 15μl 15μl
After the gel had run the proteins were transferred from the polyacrylamide gel to the PVDF membrane using a semi-dry electro-blotting protocol. The stacking gel was separated from the separating gel and discarded and the separating gel soaked in semi-dry transfer buffer solution (150mM glycine, 25mM Tris, 10% Methanol). The membrane was soaked in methanol for approximately 10secs and then in semi-dry buffer. 3 pieces of 3mm chromatography paper (5x9cm) were stacked on the electro-blotter after being briefly soaked in the buffer and air bubbles removed by rolling a round pipette across the surface. The membrane was placed on the paper and the gel on the membrane with a further three soaked pieces of chromatography paper on top. Any further air bubbles were carefully removed and the apparatus assembled and transferred for 1 hour at 300 volts.
After the transfer was completed the membrane was incubated with blocking buffer (50mM TBS-T, 10% Marvel fat free dry milk) overnight. The membrane was incubated with 4ml of blocking buffer, 8μl of the specific antibodies and 1μl of β-actin for 3 hours, rotating constantly. The membrane was washed 6 times for 5mins each in TBS-T, and incubated for two hours in blocking buffer and 2μl of specific secondary antibody (goat anti-mouse and goat anti-rabbit) rotating constantly. The membrane was washed 5 times for 5mins each in TBS-T, and then once for 5mins in PBS to remove the tween 20.
The bands were visualised using X-ray photographic film. The membrane was covered with 1ml of stable peroxide solution and 1ml of luminol / enhancer solution (both Pierce SuperSignal West Pico Chemiluminescent kit) and wrapped in colourless Saran food wrap. The light sensitive photographic film was exposed to the treated membrane in a for a period of between 30secs and 5mins, the film was then treated in Kodak developing solution for 1-2mins, briefly rinsed in water, immersed in Kodak fixer solution for 1min and washed once more in water. The film was allowed to dry overnight.