I got a really messy SDS-PAGE gel colored after transfer with Coomassie blue. I'm wondering also why the sample stopped running although the blue line with the samples was much lower than the lowest protein line in the picture (picture taken with the UVP camera where you can't see it in color).
The samples in the picture are freshly prepared rat brain samples.
As the lysis buffer I use RIPA buffer
(50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) with protease inhibitor cocktail (PIC) on ice.
After homogenising the dissected brain structure with the RIPA lysis buffer +PIC, I checked the protein concentration with the BCA method and after calculations I made sure I have 20 ug of total protein per well.
But even my beta-actin didn't give any signal at all (both primary and secondary antibody has worked fine in the past) this time.
The gel contained a 10% separating (1.5M Tris, pH 8.8) and 4% stacking gel (0.5 M Tris, pH 6.8.
As the running buffer I used the classical recipe:
250 mM Tris
1.92 M glycine
1% SDS
The electrophoresis took about 2.5 h (kinda slow!?). I set the electricity for 1 gel 20 mA.
My transfer buffer is the Bjerrum and Schaffer-Nielsen Buffer:
48mM Tris pH9.2
39 mM glycine
20% Methanol
which is apparently better for semi-dry transfer (1 h, 1mA per 1 cm2, so around 40 mA for one gel to membrane transfer).
Any suggestions in which direction I should look into?
Could be the running buffer, apparatus or gel.
1) Was the running buffer freshly prepared? Is this a continuous buffer system? As a control, borrow someone else's running buffer and run the samples as before on a gel made from the same components.
2) I assume you cast your own gels. Was the SDS and APS freshly prepared? I recommend making all new gel components i.e. both Tris solutions, Acrylamide/Bisacrylamide, APS and SDS. As a control, borrow a gel from someone else or use a commercially manufactured one, and run as before in your initial running buffer.
Assuming that you meant Methanol instead of Ethanol in your transfer solution, I doubt its a transfer issue. I would also suggest CBB staining an identical gel pre-transfer, just in case.
If, when you make these modifications, you still can't resolve your samples, the next step would be to look closely at how your sample is prepared.
Best of luck.
Is that a typo or flaw: 20% ethanol in Bjerrum and Schaffer-Nielsen Buffer (A quick Google search guided me it is 20% Methanol since I wondered Ethanol atually denature proteins!).
Have you tried running the same sample on gel and doing Coomassie staining without transfer (I am aware that the sample may be precious!). Your gel shows that the sample may have proteins lower than the detection range or it is denatured out due to alcohol?
Check the pH of separating and stacking tris buffer, this might help you.
Hello dear helpers,
1) the running buffer was freshly prepared from a 10x stock.
2) yes, I cast my own gels. The SDS wasn't freshly made, but the APS was. Okay, will prepare a new SDS next time. The pH levels for the different Tris buffers for gels were checked.
3) yes, I meant methanol. Will change this typo in the post :)
4) I guess it could be also too low protein or not enough "ion power' in the sample for the electrophoresis to run properly? But I trusted the BCA assay results!
5) I have tried doing the Coomassie blue staining without the transfer. It showed more protein of course. But why the same buffers are so inconsistent and show such an ugly gel this time? Does it look like contaminated buffer?
Thank you for your advice so far!
Hey
It seems that the problem is there in gel preparation only. You need to prepare fresh reagents and mix properly the contents before you pour them in the casting tray. I also had same issues at one time point, but then things were rescued by fresh reagent preparation. The reason behind improper running of samples is the polymerization of the gel itself. If gels are not properly allowed to polymerize, pore size varies a lot.
Good Luck.
Dear Beitnere,
Your protocol seems OK. However, if you do a transfer of a SDS-PAGE gel, first check the transferred protein pattern with a PonceauS staining of your blot. This gives you an idea of how well your proteins are separated and transferred. In addition, you can run a duplicate gel and stain this with Coomassie or silver in case you have precious samples to see how well your gel is capable to give a good separation of your sample proteins. Be aware that brain tissue contains a high amount of fat. Lipids can have a detrimental effect on protein separation, on protein transfer and will also skew binding of your primary antibody. So, before doing your antibody binding procedure, first check if your sample prep is OK, than check whether your proteins are properly separated and transferred. Success!
@Johan, thank you for the helpful advice!
Do you maybe know a better way to prepare proteins samples from a structure like hippocampus or striatum? I don't centrifugate the samples (because the sample is so precious), but leave them as they are after homogenising with RIPA and protease inhibitors for further denaturation in Laemmli buffer (5 min in 95o C).
Maybe I should centrifugate and take only the supernantant for further studies, so that there are less lipids which can skew binding?
Dear Beitnere, yes I would advice centrifugation anyway in protein sample prep. Very often you have substances that don't dissolve well in your buffer (including lipids). In worst cases this may even 'clog' your gel. Perhaps this may be one explanation for the results you showed. As a rule of thumb, the cleaner (i.e. devoid of interfering substances) your protein sample is, the better your Western blot results can be. Centrifugation of homogenized samples with a lot of lipids (e.g. brain tissue or fat tissue) will result in a 'fat cake' as upper phase in the tube, a clear interphase and a pellet. Try to get the interphase as pure as possible. You can do this e.g. by sticking a small pipet tip (or glass pasteur) through the fat cake. It is a bit tricky, but in our case it works well. Good luck!
Dear Johan, thank you very much!
I will try to make my samples cleaner by centrifugation & taking the clear interphase. I hope that the samples are not too time-sensitive as I stored the undenaturized sample in- 80 o C two months ago...
Dear Ulrika, normally should not be too much of a problem. By the way sorry for using your family name instead of your first name.
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