Phospho-Akt (Ser473), Cell Signaling Technology, 4060

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 very well with clear bands and almost no background signal.
Dilution 1:1000

This antibody is used for evaluating activity of the EGF-receptor pathway after stimulation or inhibition of EGFR in lung cancer cell lines. The antibody is very reliable. We have found that dilution in 5 % BSA is optimal.
Dilution: 1:500

I used this for a western blot of cultured hippocampal neurons stimulated with BDNF with great results.

Dilution 1:1000

I have used this antibody in stimulated 293 cells. It works great! Dilution 1:1000

I have also tested this on cultured hippocampal neurons stimulated with 1 nM of BDNF for 10 minutes. This works great to detect phosphorylation of AKT.

Dilution 1:200

This antibody was tested on 293 cells in culture. The cells were stimulated with either 0, 1, 10 or 100nM of insulin before being fixed and stained. This antibody showed high degree of staining compared to unstimulated. 10nM showed the highest staining. This antibody worked very well in detecting phosphorylation of Akt. It was also tested on primary hippocampal neurons where it showed great difference between stimulated and unstimulated.
Dilution 1:200

Excellent for detecting phosphorylated Akt in Western blotting.

We have used it for SY5Y cells stimulated with 100 ng/ml GDNF for 10 min.

It also works for reprobing of membranes.