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-   -   Problems with Protein Buffer for Western Blot (http://www.molecularstation.com/forum/basic-lab-protocols-techniques/56210-problems-protein-buffer-western-blot.html)

amazonic9 02-04-2009 11:37 AM

Problems with Protein Buffer for Western Blot
 
Hi everybody,

I hope somebody will be able to help us with this. In our lab we are experiencing difficulties with our western blots, and we think there is a problem with our Loading buffer. The thing is that we are obtaining separated bands where we should see only one band, for example with tubulin. We think there is a problem with the compactation of the sample. We have tested different buffers (I'll write the recipies at the bottom), and the first days that we use them there's no problem: we have defined bands and everything is OK. But two or three weeks after we start having the same problem.

The first buffer we tested was:

Tris pH 8 0.03 M
Glycerol 30 %
SDS 7.5 %
DTT (Dithiothreitol) 0.15 %
Bromphenol blue 0.05 %
H2O

Then we tried with the one that is given in this page (Molecular Station):
Sample Buffer (4X Laemmli Buffer)

* 2.4 ml 1 M Tris pH 6.8 (Same as upper gel buffer)
* 0.8 g SDS stock
* 4 ml 100% glycerol
* 0.01% bromophenol blue. Final Concentration is .02%
* 1 ml -mercaptoethanol (electrophoresis grade)
* 2.8 ml water

And as I said the first days everything went right, but then we get horrible western bands.

I guess maybe we are doing something wrong when we prepare this buffers, or maybe we store them in the wrong conditions.. We have tried storing them at -20C and at RT, and the problem occurs in the two cases.

Could someone give us some advice on how to prepare and store this buffers? Do we have to heat them a bit before using them? Is it normal to have foam due to the SDS? Or maybe someone thinks the problem is not on the buffer but on another thing...

Any comment will be welcome. Thanks in advance and I'm sorry if something it's not clear or English is not correct.

tgd 02-05-2009 01:59 AM

Re: Problems with Protein Buffer for Western Blot
 
Hello,

Could the extra bands be due to proteolysis? (Are they of higher or lower molecular weight?).

1. Do you boil your samples prior to loading the SDS gel? If so, do you make sure that you add *HOT* (boiling) sample buffer to the sample? If there is a protease present, the SDS in the sample buffer may facilitate proteolysis by unwinding the protein, and the idea of boiling sample buffer is to kill the protease immediately. If cold sample buffer is added and *the sample is then heated* this may give the protease almost ideal conditions before it is eventually inactivated (and it may do a lot of damage before it is killed).

2. How do you store your sample buffer? Frozen or at 4 degrees C? More importantly, do you store it in the presence of DTT and/or beta-mercaptoethanol? My experience is that the reducing agents are unstable and should be added on the day of the experiment. We store sample buffer without reducing agent at minus 20 Celcius and add DTT or beta-mercaptoethanol just before use. We then add boiling sample buffer to the samples, 'whirlimix' briefly, and continue boiling for a further 3 min. (If there is any froth (due to SDS) a quick spin in a minifuge will get rid of it).

If possible, the gel should be run immediately; otherwise store the samples at minus 80 degrees C.

3 Are there any extra bands visible on the SDS gel, or is it just on the Western?

I presume you know tubulin in an alpha-beta dimer? (Is it alpha or beta tubulin you are picking up?).

Finally, in my experience frothing due to SDS is quite normal, and makes no difference.

Good luck!

tgd

butters 02-05-2009 02:55 AM

Re: Problems with Protein Buffer for Western Blot
 
if the sample buffer is at fault usually ur molecular weight marker will have double band also

amazonic9 02-06-2009 09:54 AM

Re: Problems with Protein Buffer for Western Blot
 
Thanks for your answers.

tgd:

The bands could be due to proteolysis. The bands are always of lower molecular weight. I thought our extracts were "protected" from proteases because of the protease inhibitors that we put on our lysis buffer, but this lysis buffer is diluted when we add the Loading Buffer, so maybe you are OK and we have proteolysis problems. We add cold Loading buffer to our samples and keep them in ice till we boil them for 5 minutes. For now on, we'll do what you suggest in your post, we'll add boiling LB and then boil the samples. Can we wait till we have all the samples with LB to boil them all, or is it better to boil the samples inmediately after adding the boiling LB? If we can wait, do we keep them at RT?

Regarding storing of LB, we store the first one (the one with DTT) at -20C but with DTT already added. We keep the second one at RT without b-ME, and add this just before using it. So your recomendation is to keep them at -20C but without DTT not b-ME in each case.

When we prepare samples the day beffore running the gel, we store them at -20C. You recommend storing them at -80C, right?

The extra bands are onle seen on the Western.

We use an antibody against b-tubulin and we usually have only one defined and clean band, except when we start having the problems I explained.

Thanks very much tgd for your helpful comments.

butters:

We don't add our Loading Buffer to the molecular weight marker. It has it's own LB, it's a comercial MWM.

Thanks again!!


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