First of all your blots are great. Very clean, so your blot technique is excellent.  I don't think you have proteolysis, as you would get a bunch of bands rather than a single tight band. Where did you get the antibodies from? are you convinced that the antibodies are any good?, just because a company sells them doesn't mean they work.  We have had several antibodies we bought that are useless.  Can you get another antibody to confirm the target protein?  Also try non-phospho antibodies.  Finally, check your molecular weight markers, are you sure they are what you think they are?  I know it's frustrating but I think you have a solvable problem,  Try my suggestions and let me know what happens.

Good luck

First of all your blots are great. Very clean, so your blot technique is excellent.  I don't think you have proteolysis, as you would get a bunch of bands rather than a single tight band. Where did you get the antibodies from? are you convinced that the antibodies are any good?, just because a company sells them doesn't mean they work.  We have had several antibodies we bought that are useless.  Can you get another antibody to confirm the target protein?  Also try non-phospho antibodies.  Finally, check your molecular weight markers, are you sure they are what you think they are?  I know it's frustrating but I think you have a solvable problem,  Try my suggestions and let me know what happens.

Good luck

Thank you, Neil. That makes me feel a bit better. We buy our antibodies from cell Signaling which are usually very reliable. In the beginning of the year I was getting no signal at all, and then suddenly I got these bands but they were lower than they should be. I can't get another antibody at this stage but I have totals for both so I will try that. I'll also borrow molecular marker from another group to see if that changes anything. Thank you for your help!

Dear Toni,

The buffer solutions used to prepare the gels and later on to run them influences the electrophoretic mobility of proteins in the acrylamide gels. So I suggest check the pH of all the buffers and, if possible, prepare them yourself. I also suggest run a positive control for at least some of the antibodies to be certain of the molecular mass of the proteins of interest. Have you tried to blot with anti-actin, GAPDH, tubulin, or total Akt or Jak?

Good luck!

Jonas

Hi Jonas

Thanks for the reply. We used a running buffer from Biorad. I used my own running buffer today and my gels didn't run well. I don't do actin or GAPDH because we normalize to total protein, but it's a good idea to see where that comes out. I will do the total proteins next week as well. 

Toni, why you use 12% gels?. We use 9% and get the bands at the correct position and use the same antibody from cell signaling. My suspicion is that your molecular weights standdards are wrong. Please try to check these weights.

Good luck

Roberto

What MW marker are you using? 

We use 12% because that's the FastCast kit we purchase. It usually separates out very nicely and can be used for a range of proteins. The protein marker we use is the Gene Direx BluEye one. I tested the pH of our water this morning and it was 7.83. My TBST was 7.67. And the running buffer from biorad was 8.6, although the box said it should be 8.3. How do these pH's sound?

These pH values slightly higher are not matter of concern in my opinion. However, you should also check the pH of the buffer solutions you use to make stacking and resolving gels. Usually, for conventional SDS-PAGE gels, we use 1 M Tris buffer pH 6.8 for stacking gel and 1.5 M Tris buffer pH 8.5-8.8 for the resolving gel.

Well the kit we use to make the gels is just two components - so we add equal volumes of solution A and solution B, and then APS and TEMED, and pour the gel. But I will check what the box says about pH and then give that a check. I have put my membranes into b-Actin, so will see how that looks tomorrow. I also ran a new gel today with positive controls, and a sample boiled for longer at a lower temperature.

Hi everyone... I just want to give an update to hear your opinions.

I put one of the membranes into b-actin and got no signal at all. This is very strange, however, the antibody mix (antibody in TBST) had been used a couple of times.

I ran another gel on Monday and it looked great. I loaded some of my samples that I boiled for 5 mins at 95'C, samples that I boiled for 30 mins at 70'C, and positive and negative controls for p-Akt. I got a weak signal (left) at the lower place like before (my sample), and a strong signal (right) at the correct place for the positive control. Because I got a good signal for the positive control, this suggests that my buffers, gels and technique is fine. It must be my samples then, and we think something is preventing Akt binding to the antibody. Its not degradation because the bands are clean. I have put the membrane into T-Akt today so I will see how that looks tomorrow.

Do you use protease inhibitors when isolating your sample proteins? When the positive control gives a band in the correct position, but sample bands have a lower apparent molecular mass, proteolysis is the most likely explanation. 

Proteolysis can be very funny, I recall a protein (Mdr1) that would be cleaved into two components unless the homogenization buffer contained benzamidine on top of the usual inhibitor mix.

One other thing: It is easy to rely on kit chemistry when doing lab work. However, do yourself a favour and make sure that you can run the methods from scratch, and that you also understand how they work.

Yes, my RIPA buffer contained a protease inhibitor cocktail from Sigma Aldrich, PMSF, NaF and Sodium Orthovanadate. The base of the RIPA was Tris, NaCl, NP40, Na-deoxycholate and EDTA.

I have also made my own gels and buffers in the past but find that the kits produce cleaner bands.

If the positive control gave the band at the expected size, it means your technique and western blot reagents are fine. Therefore, the problem might be the preparation of the lysates, considering you couldn't see any actin in your samples as well. You said you use RIPA plus protease and phosphatase inhibitors, what looks fine, so I wonder what loading buffer you are mixing with your samples before boiling them... Have you considered preparing fresh samples?

 Dear Toni Leigh Goldswain , In my opinion one reason could be proteolysis as other friends above have mentioned, please try a loading control too (Actin and/or GAPDH). See where it comes also try a fresh protein marker together with the old one and compare them as well.  Antibodies seems fine to me.

Do the bands not necessarily have to be streaky/blurry for proteolysis to have taken place? I always thought that if you have clean bands then it means your proteins are still in tact. Thank you all for your responses!

regarding the positive control there are only 2 left possibilities for your problem:

dephosphorylation (doesn't explain the missing actin) or a problem with the proteins in general.

I would try to analyse total Akt or even better fresh samples.

to your question: since your AB only detects the phosphorylated residue of the protein, it could be a clear band, if the degradation of the fragment is allways the same size. (unlikely though).

1st, test your ab with some positive ctrls. Either some tumor cells lines or with commercially available cell extracts: 

• Test your ab's. For example: https://www.cellsignal.com/products/experimental-controls/akt-control-cell-extracts/9273
• or by using a specific pathway inhibitor that blocks Akt phosphorylation - your band should vanish

If that works, you know that your ab's work in general.

The next thing that comes to my mind is that:

• The molecular weight of specific proteins can be very different in different cell types, depending on cell type specifc glycosylation and isoforms. Cell signaling tests the ab's only on tumor cell lines, if you are using primary cells, the kDa could be different.

Additionally, I agree with Akash:

• you have to use a loading ctrl. With that you can determine if you have a general problem with your gel run. If the loading ctrls are running faster (lower kDa, then normal) as well, then you have a general problem.

Good Luck!

Thank you. The b-actin that I used had been used a few times by other people, so I will try to get a fresh one and try again. I will know about the total Akt tomorrow.

I am using rat heart tissue by the way...

Thank you everyone for the great advice!!!

Hi again..

I got great bands for Total Akt this morning. There were other bands at the lower molecular weight that I was getting previously, but the signal is much stronger at the correct molecular weight of 60kDa. Does this mean that my samples are not degraded? We think that something in the RIPA buffer is inhibiting or degrading the phosphorylation of Akt.

I am having a very weird problem now with my westerns. I don't get any signal at all. My film is completely blank. The same protein which used to work before does not show up at all. Even actin does not show, either. I tried changing Abs, making fresh lysate, using new ECL, even borrowing Abs from other labs that have worked for them, before also does not work for me. Very rarely, when I use the enhanced supersensitive ECL, and expose the membrane for 10 min, I get some signal (not as strong as actin can be), but it fades away immediately and never shows up again. Can someone troubleshoot my problem? So frustrating because I don't know where the problem lies

With the total akt at the right position and with strong signal, it is clear that something happened to your phosphorylation and no problem on the technical side. 

I would check if the lower bands are 'known' unspecific bands (website of manufacturer, own old plots) because I think that is the most likely now.

I think you have made some mistake in your question. Your blot shows the p-Akt and p-Jak bands and what is missing is the SOD1 bands. The results are right. The pJak2 band must be 11kD band. 

According to the antibody data sheet, phospho-jak2 is 125 kDa, not 11 kDa. I have not shown an image of the SOD1 bands.

Thank you everyone for all of the advice.

Toni Leigh Goldswain

I am having the same issue as you. I am working on rat heart protein extract trying to detect JAK2 and phosphoJAK2. My bands are significantly lower than expected; According to the Abcam Antibodies datasheet I am using, the bands JAK2and Phospho Jak2 should be at 120KDa. But I do not see a band at this molecular weight. Actually I observe 3 bands for both Abs: 1 band at 100KDa, another at 70kda and a third band at ~45Kda.

Could you please share how you solve your troubleshoot?

@Prisca: That sounds like degradation by a protease. Sometimes standard protease inhibitor mixes are not enough, I once had a case where the target protein was kept intact only in the presence of benzamidine, allegedly a reversible Ser-protease inhibitor. But Ser-proteases should have been killed already by PMSF, so I still don't know what was going on there.

@Engelbert, thank you for your suggestion. I am using proteases and phosphatase inhibitors coktails from Roche (PhoStop and COmplete Ultra tablets). The other thing is that with exactly the same protein extract I was able to detect STAT3 and PhosphoSTAT3 at the right molecular weight. So I am not sure if the proteases left active can selectively degraded JAK2 and leave STAT3 intact. May be I should try another type of inhibitors. It's puzzling..