Hi, I am trying to do a Western Blot (Akt1, phospho Akt1, Akt2, phospho Akt2) from HepG2 (human liver cancer cell line) proteins stimulated with insulin. Akt1 is detected, but not the other Akt isomers. I was wondering if there is something wrong with my method, or if it's the HepG2 that is responding with low levels of p-Akt1, p-Akt2, and Akt2. I will mention the significant steps of my western protocol:
I lyse the cells with RIPA buffer (don't wash with PBS) and sonicate the samples. I spin the samples, mix with proper amount of Lammeli+B-ME buffer and load 40ug of proteins in the gel (7.5%) then run for 200V for 30min. I use semi-dry transfer (20V for 30min) and then use the Odyssey TBS blocking buffer to block the membrane and use 1X TBS for washes. I incubate the membrane in primary antibodies (1:1000; from Cell Signaling Technology) overnight in 4C and in secondary (1:2000) for 1hr in room temp.
Is there a special precaution that must be taken to detect phospho-proteins? Thank you very much!
Dear Jaewon,
Have you included protease and phosphatase inhibitors to prevent degradation and dephosphorylation of targeted Akt proteins? It is very important. If not, use protease and phosphatase inhibitor cocktails from Thermo, Roche or Sigma.
It might be also related with a bad quality of antibodies. Besides, I'm not familiar with HepG2, but the abundance of 3 Akt isomers is variable in different cell lines. Some providers have useful information about expression of signaling protein such as Akt in different cells. Look through web sites of anti-Akt antibody providers.
Below, I also suggest the article in which Akt1 and Akt2 expression/phosphorylation were studied. Good WB results.
https://www.researchgate.net/publication/24180753_PTEN_Contributes_to_Profound_PI3KAkt_Signaling_Pathway_Deregulation_in_Dystrophin-Deficient_Dog_Muscle
Article PTEN Contributes to Profound PI3K/Akt Signaling Pathway Dere...
1. Isoform expressions are cell line dependent.
2. 40ug is a bit high: after boiling, the samples form aggregates in b-ME+Lam. buffer. So, there is a chance some proteins stuck in the wells. Try loading 20ug, 30ug and 40ug in same gel to check this out.
3. Use new stock for antibody. and include a positive control.
1. Usually primary western antibodies from CST are recommended to be diluted in either milk or BSA in TBST, so be sure to check which one is best for your antibodies [Your antibodies and washes should be made up in TBST (TBS + tween) and not just TBS]
2. Because your protein is so concentrated, you might need to add a greater concentration of protease and phosphatase inhibitors to see phospho proteins
3. If you antibody has weak signal, usually a wet transfer is better than a dry transfer
Good luck!
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