Hello everyone,
I am trying to probe for phosphorylated ERKthrough western blot. How different is the protocol from regular western blot? I looked up on abcam and they ask to use bsa instead of milk for blocking. Other than that is there any other precautions to be taken? Is it advisable to store the samples in -80 or in -20after boiling with blue loading buffer? Kindly advise on how to go about this experiment, I tried twice with no success and this is very crucial for my project. Thank you so much.
sincerely,
Lakshmi.
8 Tips for Detecting Phosphorylated Proteins by Western Blot
https://advansta.com/8-tips-for-detecting-phosphorylated-proteins-by-western-blot/
DO NOT USE ALKALINE PHOSPHATASE FOR DETECTION!
Check the answers in a previous thread:
https://www.researchgate.net/post/How_can_I_get_phospho-protein_signal_in_Western_Blot
Also have a look at this paper:
http://onlinelibrary.wiley.com/doi/10.1111/sms.12702/full
It's not sufficient to use only protease inhibitors in preparing your samples, so be sure to use both protease inhibitors AND phosphatase inhibitors.
Storing samples at -80C after boiling is always a good idea.
BSA instead of Milk is also advisable, as is TBS(T) instead of PBS(T).
Yes, the main point is to avoid dephosphorylation - phosphatase inhibitors should be added already during cell lysis, and samples kept on ice,as detailed in the abcam protocol.
http://www.abcam.com/ps/pdf/protocols/western%20blotting%20of%20phospho-proteins%20protocol.pdf
Milk contains casein, which is a phosphoprotein and can cause a high background. Phosphate in the buffer can competitively inhibit the binding of phosphorylation specific antibodies.
8 Tips for Detecting Phosphorylated Proteins by Western Blot
https://advansta.com/8-tips-for-detecting-phosphorylated-proteins-by-western-blot/
DO NOT USE ALKALINE PHOSPHATASE FOR DETECTION!
Check the answers in a previous thread:
https://www.researchgate.net/post/How_can_I_get_phospho-protein_signal_in_Western_Blot
Also have a look at this paper:
http://onlinelibrary.wiley.com/doi/10.1111/sms.12702/full
Hi, for detecting phosphorylation you have to add phosphatase inhibitor in your lysis buffer. I add sodium vandate (1mm)in my lysis buffer. It is good to use washing buffer along with sodium fluoride that helps in getting good results.
ERK phosphorylation is quite convenient to detect, just take care of timing. You should be able to see phosphorylation somewhere from 15 min onwards.
Once your cells are 70-80% confluent you should be ready to give treatment or your experimental condition. Once the experiment is over discard the media and give a quick wash with pbs. (yu can lyse the cells in the plate only). After PBS wash keep your plate immediately on ice and to it add ice chilled RIPA buffer along with protease inhibitor as well as phosphatase inhibitor. Keep on shaking for approx 20 -30 min followed by vigorous pipetting and scraping the lysate. Transfer the lysate in a fresh Eppendorf and centrifuge at high-speed at 4oC rid of the debris.
I will advise to run the gel and blot the same day of lysate preparation to get best result. Once the protein is transferred on the membrane, block the membrane with 2.5%BSA-PBS for 1-2 hr at 370c or 40C O/N. Incubate with primary antibodies ant-pERK preferably overnight and prepare the antibody dilution in 1%BSA-PBS. CST antibodies works best and the antibodies can be used multiples time.
Give 3-PBST or TBST (with NaF) washes 5 min each with shaking. Incubate with secondary antibody (prepared in 1% BSA-PBS) for 1 hr. Give 3-4 washes with PBST-TBST followed by one final wash with PBS.
Image your blot immediately if using HRP based secondary.
Once phosphorylation is detected then you can re-probe with ERK antibody to detect total ERK.
Good luck
Thank you so much for your suggestions, Dr. Tiwari. I used 2 different cell lines BXPC-3 and PANC-1 for this experiment. I treated the cells for 24 hrs with the inhibitor and prepared the cell lysates in ice. I treated both cell lines under same conditions, but I am able to detect pERK in PANC-1 and not on BXPC-3 (KRas WT). Even the untreated BXPC-3 sample doesn't show pERK. I am not able to explain this. Kindly help me trouble shoot this, I would be deeply thankfull for any suggestions.
Thank you,
Sincerely,
Lakshmi.
Hi Lakshmi,
Some cell line needs treatment with some signaling activator like tgf beta etc to activate the phosphorylation to take place. You can see which is the relevant treatment necessary in your experimental setup to activate ERK phosphorylation. If you need to discuss in detail about experiment please drop your number in message box, I can call on Saturday to discuss the experiment.
Good luck,
Anil
I would add the use a high sensible chemiluminescence substrate like femto.
Hello Dr. Valenzuela,
Yes, I used super signal chemiluminescent substrate from Thermo Fischer. I got a strong pERK signal for PANC-1 samples though BXPC-3 didn't show any. Both samples were prepared and run on the same gel.
Thanks,
Lakshmi.
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