Weak Western band for phosphorylated Akt - whats happening?? (Oct/26/2005 )

Hi Guys,
I have been doing westerns with P-akt for a while now. Earlier I used to sonicate my cells and now I have switched to cell lysis without sonication.

I am not sure but these days my p-akt band is very light even after an hour exposure...is it due to the way I am harvesting my cell lysate?? Also my antibody that I am using was bought in August...

Another variable I am not sure though...is of the protein.. I used to load 25 ug (since with sonication my yield used to be low) but after i switche dto the cell lysis (since my protein yield is higher with this) I load 50 ug... Is this a factor...

This might sound weird but today I had two gels one had 30 ug and the other had 40 ug protien...I saw a couple of bands in the 30 ug one and not the 40 ug one....therfore my question is:

1. Is it the sonication
2. Is it the antibody
3. Is it the amount of protein that I load...

hi there,
did you perform a bradford or lowry to determine protein content after lysis or sonication? you might want to do this as this will give you a rough idea of which method, lysis or sonication, yields the higher protein content. it would also give you a rough guide as to how much you must load for your gels.

i do a bradford and thats how i figured that my homogenization was givng mw a higher protein than sonication....

One thing i need to add is my T-akt signal is comparable by both methods its jst the p-akt...

sorry guess i eating up letters...just too hungry :-)

Hi There Pria-

I wanted to borrow some P-Akt (ser473) antibody from a postdoc in another lab and she told me that it often gives very weak signals and that they really had to optimize it to get it to work well at all. I'm not sure if you are using the same antibody, but this may suggest that your problem may be with the antibody. The antibody that they gave me was from Cell Signaling #9271.

When they gave me the antibody they also gave me a brief protocol.

It was:

load ~20ug run out and transfer to PVDF
block 30 mins in 5% milk
wash with TBST (only 0.1% tween for all washes)
incubate membrane with pAkt Ab 1:1000 in 5%BSA overnight (they said it won't work in milk)
wash 3x5 mins with TBST (Don't over wash)
add secondary (whatever yours is)
wash 3x5 mins and develop with your system

Hopefully this helps you. If you think your problem may be with the antibody, it might be worth trying their protocol.

good Luck

Mountainman

i use 4501.....i think the antibody might be different since the batch is different...anyway i am using a very similar protocol except that the antibody is in milk...they recoommend that. I used to get really storng bands with this antibody...now it not too strong...

Happy to know that someone agrees with me.. thanks a lot for the reassurance.

It may be batch-to-batch variability, although unlikely. Does the lysis buffer contains any phosphatase inhibitor(s)?

si....it does

i add naf, NaVanadate, NaPpi and protease inhibitor cocktail (six protease inhibitors) from calbiochem..

Irinically earlier i used to use a ripa buffer which had only apro, pepst, PMSF, leupep and na vandate.

Hello,
I had huge problems while doing westerns to detect P-Akt. A big part of my time went to optimise the system.
First I used Cell Signallings antibody, and it was really crappy. It gets bad fast so if you have an old antibody you might want to get a new one. And when you get it you can aliquote it so you avoid condensention in the tube everytime you take some ab. I got really bad results with milk in my TBS-T. It preferres BSA. The antibody for total AKT is very good and I never had any problems with it.
Now did you use TBS-T? PSB-T can interact with phosphorylated proteins.
Another thing is that P-Akt seems to be really unstable. After Lysis in RIPA+phosphatase inh.+ protease inh. I collected the supernatant and did a bradford assay = directly after that I mixed the supernatant with Laemlli. It doesn't like to be freezed. I also used 6X Laemlli so you might try to increase your SDS concentration if you have a lot of protein. I kept this in the fridge and loaded gels the next day.
I got a much better transfer of Akt with wet-transfer than with semi-dry transfer. And never color your membrane with ponceau, it distroyed my akt totally. Use a prestained marker instead.
Maybe you do all this already but I thought I would share my tricks as I know how frustrating P-AKT can be!
Good luck

Hi mickihiiri,

I have still not solved the problem :-) every week seems to be giving me different results. the answers to ur questions:

I use TTBS and I use fresh sample every time. I do not reuse samples for P-akt. I use milk since it says milk in their brochure...T-akt has never been a problem. I use a wet transfer system and never stain my membrane with ponceau or anything...

This interesting thing happened to me last week and I am still figuring it out. I would love to see what u think.

I ran a western last monday and I did not get any signal in that membrane. I ran some other sample on Tuesday and resused the antibody dilution (antibody+ milk) and I got wonderful bands on Wednesday within 5 mins of exposure. The only thing different in both the days were that I made fresh transfer buffer (yes It does not make any sense) on Tuesday. I reused the same antibody dilution 4 more times (i did not add any fresh antibody) and I got a signal on all the days.

Yesterday I ran another western and I decided to use a fresh antibody dilution and no I did not get any signal. The antibody was from the same batch and vial...The cells were same and everything else was the same. Transfer buffer was last weeks...I am not sure if this h as anything to do. Therefore, I am running another western today and I have made fresh transfer buffer today. lets see if this is the variable..... What do u think is going on???

Hello again!

Indeed it sounds very strange. Was it sample from the same cell batch on monday and tuesday?
I was never able to reuse my p-akt ab-solution it got so bad already the next day so at least youre lucky with that. But I never needed to expose for as long as 5 minutes after I had found the protocol that worked.

I didn't quite understand about the transfer solution though. What do you mean by fresh? Do you put methanol in it when you prepare it? The methanol should be added to the buffer the same day as you run the transfer and cooled down before use. Maybe you find me silly to ask but its just to be sure if the problem could just be there.

Is it so that you run other westerns as well? and have no problems? I suggest that you run a positive control in your western (if you can find insulin for example to treat the cells with, that should give you a huge amount of p-akt). That way you will know if there is a problem with the samples or the westerns. But I highly suspect that there is something in the western protocol that the p-akt do not like, or maybe in the lysis. When you say you got no bands, did you check if you had total akt bands anyway? How long do you transfer? I transfered at 400mA for 2 hours.

I will work with another protein now and I actually just e-mail cell signaling to ask what they suggest for that particular one. Maybe you could do the same to your company?

I run tissue samples to find p-akt so I lysed with RIPA buffert (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0,25% Na-deoxycholate) . I added Mini Complete Protease Inhibitor and 1 mM NaF and 1 mM NaVO just before use. I guess you have activated your NaVO as required. I homogenised with a needle, left it on ice for 30 min for dissolvation and centrifuge in +4C for 45 min. I added 6X Laemlli buffer 1:1. It might be useful to have a higher concentration SDS if you have a high amount of proteins. I loaded 30 microgram protein to the wells.

I have no idea if this helps you at all, but I hope you will resolve this problem soon.