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Thank you for getting back to me!
Yes, we did see other size bands. I’ve scanned in the blot and attached it for you to see.
I loaded 100ug of protein (I know...Lots!).
We have a lysis buffer we prepare in our lab:
20mM Tris/HCL, pH 7.5
20mM Sodium Fluoride
1mM Sodium Orthovanadate
2mM Sodium Pyrophosphate
4. We did the western blot twice.
The first time:
primary antibody at 1:5000 in 5% milk, incubated overnight at 4 deg C. secondary was Goat anti-Rabbit HRP from Jackson Immuno at 1:10,000, in 5% milk, 1 hr at RT.
The second time:
Primary antibody at 1:1000 in 5% BSA, incubated overnight at 4 deg C. secondary was Goat anti-Rabbit HRP from Jackson Immuno at 1:5,000, in 5% milk, 1 hr at RT.
Asked on Jan 18 2013
Thank you for the additional protocol details and for the image provided. Have you tried any shorter incubations with the primary, such as 1 hr? Although we normally recommend ON for the strongest signal, reducing this time may eliminate some of the background bands. GIven that a Western Blot band can vary between samples and due to things like buffers up to 10 kD, it looks to me like the band around 60 kD may be your P70 S6 kinase beta. We do show some faint additional bands on the blot on our datasheet with a note indicating that we are unsure of the identity of these bands.
We do guarantee our products within 6 months of purchase and can replace, credit, or refund if this antibody does not work successfully for you.
Answered on Jan 18 2013