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I have a problem in western blot that I can't resolve by myself. When I am use to add 100 microgram of proteins for each sample but after running and transfer its impossible to me to see bands of protein. What can be the reason of that? Thank you for your helpenter image description here

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    $\begingroup$ What kind of protein are you using: Whole cell extracts or purified samples? What are your running and transfer conditions? Have you tried to stain the gel with coomassie blue to make sure you have sample on the gel? How do you measure your proteins? What are the buffers in which you measure? $\endgroup$ – Chris Mar 26 '15 at 13:01
  • $\begingroup$ so I need to quantify globin which is protein of 16 kda. I extracted my proteins from MEL cells after treatment by using buffer lysis and protein inhibitors. I do the running at 80volt in running buffer ( tris,glycine) and then I do a wet transfer for an hour with transfer buffer ( tris, glycine, methanol). I didn't think to stain the gel but it's actually a good idea that will proof if my problem in the running or transfer step... To measure my protein I am doing Bradford method when my Lysats are in 200 micro liter buffer lysis . $\endgroup$ – Anael Mar 26 '15 at 13:24
  • $\begingroup$ Did this blot ever work before? What is in your lysis buffer, by some chance SDS or something like DTT? $\endgroup$ – Chris Mar 26 '15 at 13:25
  • $\begingroup$ I was first using a tris-triton buffer lysis then I use a buffer lysis that contain tris, triton, NaCl,EDTA and . I try to to use in an other blot and same result: I can see proteins band in the upper side only... $\endgroup$ – Anael Mar 26 '15 at 13:36
  • $\begingroup$ What do you mean by seeing proteins only in the upper side? $\endgroup$ – Chris Mar 26 '15 at 13:38
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From what you say, I can only give a few advices:

  1. For the quantification, make sure your sample does not contain any substances which disturb the assay. The Bradford assay is for example sensitive against SDS or DTT. See here for more details. This prevents you from loading a sample without enough protein.
  2. Make sure that you really have protein on your gel. Run a gel as normal and then stain it with Coomassie Blue (it cannot be used for western afterwards).
  3. Don't loose your sample - this happens easier than most people think.
  4. Make sure your transfer works. This can either be done by using pre-stained ladders (which are helpful anyway) or by doing Ponceau Red staining. This can be washed off with water and doesn't disturb downstream applications.
  5. Depending on the type of membrane you are using, they might need pre-treatment. PVDF membranes are extremely hydrophobic and need to be activated in methanol.
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  • $\begingroup$ Thank you I will check for the buffer lysis . How can I be sure that I will not just loose the proteins? $\endgroup$ – Anael Mar 26 '15 at 14:16
  • $\begingroup$ Hi Chris, i have joined to the question a picture of my membrane after ponceau if you can take a look. $\endgroup$ – Anael Mar 27 '15 at 13:20
  • $\begingroup$ I will have a closer look later. Can you tell me the size of the marker (bottom 4 or 5 bands are enough). $\endgroup$ – Chris Mar 27 '15 at 13:29
  • $\begingroup$ It's a 10-250kd marker of 10 band $\endgroup$ – Anael Mar 27 '15 at 14:14
  • $\begingroup$ I don't see any bands here, but only a smear. I would first suspect degradation of your sample. It would be really important to simply run a gel and stain it to see how this looks like and to find out the problematic step here. It's probably already the gel... $\endgroup$ – Chris Mar 27 '15 at 21:18
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After I had check so many possibilities I finally resolve the problem. It turns out that ph of the tris 1,5M for gel preparation was too many basic...which explain a smear of the proteins into the gel

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