The membrane looks very bad.

1. Try to use nitrocellulose membrane.

2. Why are you probing the same membrane with 6 antibodies when size of all the proteins are same? You  can run same sample for 6 proteins in 2 gels and cutting the membrane and incubating the membrane with each antibodies separately.

Dont probe same membrane with 6 antibodies when you have all proteins of same size.

Also use 5% non-fat dry milk. When you transfer the membrane, do wet transfer to avoid bubbles problem.

Wash membrane 3 times, 15min each with 0.1%TBST on room temperature.

Try to use better primary antibodies (monoclonal) to reduce non specific bands.

I hope you will get better results this time.

Suggestions made by Sandeep are good options I think.

Additionally, you have another important factor in your experiment which is your lysis buffer. Which detergent did you use and have you tried alternatives? Besides widely used Triton-X, NP40 might be a good alternative for you.

Accordingly, the amount of lysis buffer and tissue used also plays a central role for lysis efficiency. It might also help to use physical disruption of your tissue prior to lysis in order to improve the protein yield.

Also, since you want to look at phosphorylation, did you add phosphatase inhibitors to your lysis buffer? Did you add protease inhibitor?

When you say you are getting the exact same 4 bands with each antibody, your secondary antibody might be the problem. You could try and titrate it to obtain better results.

Finally, are you sure all your antibodies recognize the Drosophila protein? I just quickly checked the cell signaling website and the yap antibody that they offer for example is not listed to recognize Drosophila yap.

Hope this helps and you can improve your WB.

Hi, all the above suggestions are really good, I can only say that I worked with Drosophila so first of all check the datasheet of the antibody, as Martin said, for this reason, point 1), there is a possibility that you see non specific band.

It is better that you run more than one gel. You have to probe p-JNK and p-Yap, get results at same weight, then stripped the membrane for the total proteins (JNK and Yap) and again stripped the membrane for the tubulin always at 50KDa (maybe you can choose an antibody, at a different weight, for normalization!?), it is not simple! Do you have a positive and negative control for phosphorylation activity? You have to be sure that you see an activity, so you can control also if your band are specific or not.

For point 3) sometimes this streaks were caused by precipitation of you samples between the stacking and running gel (I suppose that you used a hand made gel), how do you homogenate the samples? Do you use the detergent suggested by Martin? If you are sure of your lysate you can vortex your sample before and after boiling at 95°C, and pay attention to not let dry the running before adding the stacking gel.

Good luck and go with the... Fly!!

One more suggestion, you can increase the dilution of antibodies or reduce the incubation time.

vertical lines in the bands may represented that your have some problems in protein dissolving, make sure your lysis buffer is strong enough to dissolve proteins in your targetting tissue.  good luck!

I used to have the same probles for p-JNK. I was using antibodies from Santa Cruz. My solutions were:

- Block 1 hour in BFS (Bovine fetal serum diluted 1:10 in PBS)

- Reduce primary antibody dilution to 1:1250

- Reduce secondary antibody dilution to 1:55000

- For protein  visualization I used Immobilion Western chemiluminescent HRP substrate (Millipore Corporation, Billerica, MA, USA), but if you have a high background you can use Pierce chemiluminescent kit, since it is less sensitive.

Hope it works for you

I never see this suggestion anywhere, but I have found that it is important if you want the cleanest background. Some of the background seen on your blot could be from a failure to wash the BACK side of the blot and to remove antibody "stuck" between the blot and the dish.  Try flipping the blot over for at least a few minutes of at least one of the three washes, after both the primary and secondary antibodies.  A rocker, rather than a circular washer, gives more even washing. Also, make sure that when you incubate, rock or wash that the solution or buffer is level across the blot and that there are no bubbles under the blot. Bubbles can lift the blot out of the solution enough to create artifactual lack of staining later. As for the stripping, be sure to block again afterwards to "re-seal" it against non-specific antibody adherence.  Hope this helps!