I've tried the rabbit anti-pCREB from Cell Signalling and had very low band intensity, in contrast to anti-CREB which works well. The rabbit anti-pCREB from Millipore works much better (cat no 06-519) - I use it at a 1:1000 dilution in 5% milk blocking buffer, incubate overnight at 4C.

A good detection of pCREB depends on which tissue you work in (in pineal gland and brain pCREB is generally easy to detect, other tissues are less suitable), the antibody you use is extremely important. We worked from the mid-90ies to some years ago with Cell Signalling rabbit ant pCREB, which became bad, then we used one from Millipore now Merck Millpore and these days I work with a rabbit monoclonal from CS again, 87G3 at 1:1000 dilution in brain tissue and neuronal cells/cell lines. To really be able to help you I need some more details. Our current Western procedure is described here: Benz et al., 2010 British Journal of Pharmacology, for pCREB in Maronde et al 1999 Journal of Neuroscience or Fricke et al, Endocrinology 2004 and 2005).

Cell signalling have very bad antibodies for p-CREB a simple trick is use FEMTO ECL and the dilution of Antibody 1:500 in BSA 3% , transfer 250 mA without SDS, (running GEL 12 %)

I've tried the rabbit anti-pCREB from Cell Signalling and had very low band intensity, in contrast to anti-CREB which works well. The rabbit anti-pCREB from Millipore works much better (cat no 06-519) - I use it at a 1:1000 dilution in 5% milk blocking buffer, incubate overnight at 4C.

Thank you for the suggestions. Am actually working with brain tissue (Hippocampus and Frontal cortex) which should be easy to detect!

I value and will consider all the contributions.

Erik,

Could you please help me with more details of the literature you referred me to? i.e "Maronde et al 1999 Journal of Neuroscience or Fricke et al, Endocrinology 2004 and 2005)" May be the journal titles or the URL where I can get them.

Thanx

When you send me your E-Mail adress, I can mail you all of them. My business Mail-adress is [email protected] the private one is [email protected]. Best, Erik

Stephem,

I just saw that you can get the pdfs also from my publication list here on ResearchGate.

Thanks

Pablo Andrés Muñoz Llancao,

I would like to know how long when you tranfer 250mA? Thank you very much.