Phospho-CREB, Cell Signaling Technology, 9198

We have used this antibody to detect phospho-CREB in primary mouse hippocampal neurons and in mouse hippocampus homogenates.

I have tested this antibody on mouse HepG2 cells, stimulated with 10nM insulin for 10 minutes.
The cells were lysed in kinexus lysis buffer (see below) and 50ug protein were loaded in each well for western blot.
Dilution: 1:1000
The membrane was left to incubate over night at 4 C in blocking buffer (milk).
The antibody worked very well.

Lysis buffer:

20 mM MOPS, pH 7.0 (any other buffer at this pH could be substituted);
• 2 mM EGTA (to bind calcium);
• 5 mM EDTA (to bind magnesium and manganese);
• 30 mM sodium fluoride (to inhibit protein-serine phosphatases);
• 60 mM β-glycerophosphate, pH 7.2 (to inhibit protein-serine phosphatases);
• 20 mM sodium pyrophosphate (to inhibit protein-serine phosphatases);
• 1 mM sodium orthovanadate (to inhibit protein-tyrosine phosphatases);
• 1% Triton X-100 (can be substituted with 1% Nonidet P-40) Important Note: Do not add if you intend to first prepare a cytosolic fraction.
• 1 mM phenylmethylsulfonylfluoride (to inhibit proteases);
• 3 mM benzamidine (to inhibit proteases);
• 5 µM pepstatin A (to inhibit proteases);
• 10 µM leupeptin (to inhibit proteases);
• 1 mM dithiothreitol (to reduce disulphide linkages)

adjust to pH 7.2

This antibody works great in both WB and immunostaining.
The antibody was used on primary cultured murine neurons.
WB dilution: 1:1000
Immunostaining dilution: 1:800

I have tested this lysates from mouse hippocampal neurons with great results!

The neurons were stimulated with 1nM BDNF for 10 minutes before lysis in lysis-buffer containing phos-stop and complete.

50ug protein was loaded per sample. The antibody was used at a 1:1000 dilution and left to incubate over night at 4 degrees.

A clear signal was observed in the sample stimulated with BDNF while a very weak band was observed in the unstimulated sample.

I have used this antibody with great results for western blotting of lysates from cultured murine hippocampal neurons. It gives one specific band at 45 kDa when loading 50 ug sample and incubating in a dilution of 1:1000 in blotting buffer at +4 degrees ON.
I have resumed this AB twice.

Phospho-CREB (Ser133) (87G3) Rabbit mAb #9198 from Cell Signaling

Gives one specific band at around 45 kDa
I have used it for western blotting with 20 ug WCE of primary mouse hippocampal neurons and with 20 ug WCE of the human neuroblastoma cell line SH-SY5Y in the dilution 1:1000.
Incubation at +4C ON. Can be reused 3 times.