Phospho-MAPK, Cell Signaling Technology, 4370

Great antibody for IF. Concentration indicated by manufacturer’s protocol.

For more information see Micali et al. Cell Reports 2020, “Variation of Human Neural Stem Cells Generating Organizer States In Vitro before Committing to Cortical Excitatory or Inhibitory Neuronal Fates”.
https://www.sciencedirect.com/science/article/pii/S2211124720305489

WB dilution 1:2000

IF rating: 5 stars
IHC rating: 4 stars

For more information see Sutton et al. Cell Reports 2019, “DIRAS3 (ARHI) Blocks RAS/MAPK Signaling by Binding Directly to RAS and Disrupting RAS Clusters”
https://www.sciencedirect.com/science/article/pii/S221112471931527X#sec5

This antibody worked well with lysate of murine cortical neurons. The neurons were lysed with Kinexus lysis buffer. Clear bands were observed after 1 min. exposure.
Diltution 1:1000

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

PHOSPHO-MAPK (ERK1/2) (THR202/TYR204) RABBIT MAB CELL SIGNALING #4370

I have used this antibody several times for western blotting of primary hippocampal neurons stimulated with BDNF. Blots have always been succesful. It detects two distinct bands at 44 and 42 kDa. There are very little or no unspecific bands to be detected.

I use this antibody routinely for western blotting of PC12 cell lysates or NSC34 cell lysates following growth factor stimulation (NGF/BDNF). It works brilliantly at 1:2000 on as little as 1µg of protein per lane, blocked with 3% BSA in TBST.

I used this for Western blotting, it worked well at a 1:2000 dilution on neuroblastoma cell lysates, following BDNF treatment of live cells

Detects pERK levels (kDA 42,44) very nicely in WB (dilution 1:2000, 5%BSA/TBS/Tween).
Have tested it on lysate from MG87 cell-line as well as on embryonic mouse kidney total lysate (E14).

Excellent antibody for Western blotting.
We stimulated SY5Y neuroblastoma cells transfected with human TrkB with increasing concentrations of BDNF for 15 min. Cells were lysed in TNE buffer containing protease and phosphatase inhibitors. Lysates were analyzed by Western blotting and blots were developed using ECL Plus.
We observed a dose-dependent response to BDNF using this antibody. Two intense band around 50 kDa were induced by BDNF.
WB 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 MAPK.

Dilution 1:200

I tested this antibody in 293 cell cultures which had been stimulated with 10nM insulin. It worked excellent in detecting insulin-induced phosphorylation of MAPK using Western blot. I diluted it 1:2000

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 Mapk. It was also tested on primary hippocampal neurons where it showed great difference between stimulated and unstimulated.
Dilution 1:200