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Markers hardly dissapeared


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#1 Irene G

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Posted 13 March 2009 - 03:56 AM

Hi everybody!
We are having problems with our western blots. The last thing we have is that after a good transfer (25 mM Tris, 190 mM Gly, 0,1% SDS and 10% methanol for searching proteins of 120 KDa), with all bands of markers ok (prestained and kaleidoscope, both from BioRad) we have blocked with 5% milk (we have bought one with calcium and bifidus, don't know if there is a problem) in TBST and the markers are almost dissapeared. Has anybody idea of what has happened? We have continued with the blot and it has a big background and we can't see the good bands. Now we are staining with ponceau for watching the look of the proteins. Some advice please!

#2 rkay447

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Posted 13 March 2009 - 04:30 AM

Couple ideas. First, all transfer buffer recipes I've ever seen has 20% methanol. I know the methanol is a fixative which fixes the proteins to the membrane. Perhaps your proteins are not getting fixed well enough and are coming off the membrane easily.

The other problem might be in the TBST/milk. It sounds almost as if the membrane is being stripped. Check the pH of your TBST and then test the pH with the milk. I'm wondering if your milk is the problem. Why are you using a milk with bifidus? You know this is bacteria, right? I'm not sure if your milk actually has the bacteria (does the box say with probiotics?) or a bifidus factor which greatly encourages the growth of the bacteria (does the box say with prebiotics?). Either way, this could decrease the pH quickly. This can strip a membrane and destroy antibodies pretty quickly as well. I'm not sure if this is truly your source of problem but why introduce an unknown and potentially destructive experimental condition with your milk? Really, go to the local market and get a great big box of plain and simple dried, skim milk. Why take the risk and potentially destroy experiments with an odd milk?

These problems are clearly not exclusive and you could be having a combination of the two which would most certainly destroy your membrane.

#3 klinmed

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Posted 13 March 2009 - 05:16 AM

Hi everybody!
We are having problems with our western blots. The last thing we have is that after a good transfer (25 mM Tris, 190 mM Gly, 0,1% SDS and 10% methanol for searching proteins of 120 KDa), with all bands of markers ok (prestained and kaleidoscope, both from BioRad) we have blocked with 5% milk (we have bought one with calcium and bifidus, don't know if there is a problem) in TBST and the markers are almost dissapeared. Has anybody idea of what has happened? We have continued with the blot and it has a big background and we can't see the good bands. Now we are staining with ponceau for watching the look of the proteins. Some advice please!


We use 20% methanol in the buffer, but if transfer was ok donīt think that this is the problem. With nitrocellulose we completely air-dry the blot before blocking (usually storing overnight in a dissicator at 4oC). This seems to greatly facilitate immobilization. We then wash briefly in TBS before blocking. Your milk seems a bit fancy! Most people use plain skim milk powder.

Hope this helps.

#4 little mouse

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Posted 13 March 2009 - 06:01 AM

I also had a problem with a kaleidoscop marker (don't remember the provider).
the colors are fading.
My advice, use a pen to label again the marker on the blot. Do it right after the transfer. I used a blue BIC (the black one is not OK, because the color is slightly spreading on the blot after a while of agitation).

For the background : follow the good advices of klinmed and rkay. change your milk. Be careful, prepare it fresh as it can be contaminated very fast, with pH decrease that can lead to background (I personnally experienced it)

Edited by little mouse, 13 March 2009 - 06:05 AM.


#5 sciencedork

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Posted 13 March 2009 - 11:14 AM

Markers do that sometimes. It is my personal opinion that you need to check how much TWEEN you are using. TWEEN at a 0.1 % or higher can cause proteins to degrade (and therefore, MW bands to disappear). I now use 0.05% Tween or no Tween at all if it will be stored for a couple of days (over the weekend blocking, etc...). Lastly someone mentioned using a marker to mark the molecular weight bands before blotting. Great idea and I do it myself. Takes the worry out of what happens to the markers all together. The only problem I have with markers are the See-Blue which have one red band and all the rest are blue. The red band always disappears. I have never given it much thought since it is marked by me before continuing to the next step. Just be sure the blot does not dry out while marking the bands. You will need to blot off the area where the markers are so the pen does not spread out, but do not blot the rest of the membrane. Leave it wet.

Edited by sciencedork, 13 March 2009 - 11:19 AM.


#6 sciencedork

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Posted 13 March 2009 - 11:18 AM

I forgot to mention about the milk. Use the cheap brand...Roundy's whatever you have. Do not use the name brand Carnation. I have heard there are issues with that brand being used for westerns. Non-fat skim milk is all you need. Do not use additives. You can use either TBST or just TBS for blocking. Best of luck to you.

Edited by sciencedork, 13 March 2009 - 11:20 AM.


#7 lotus

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Posted 13 March 2009 - 11:44 AM

I forgot to mention about the milk. Use the cheap brand...Roundy's whatever you have. Do not use the name brand Carnation. I have heard there are issues with that brand being used for westerns. Non-fat skim milk is all you need. Do not use additives. You can use either TBST or just TBS for blocking. Best of luck to you.


I have had problems in the past with making a lot of 5% milk and storing it for a long time. The milk tends to degrade and this ruins the blot. So make your milk fresh every time and throw away any excess you have. After all, skim milk powder is cheap!

otherwise, buy a commercial blocking powder like Blotto from santa Cruz.

#8 klinmed

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Posted 13 March 2009 - 12:38 PM

Markers do that sometimes. It is my personal opinion that you need to check how much TWEEN you are using. TWEEN at a 0.1 % or higher can cause proteins to degrade (and therefore, MW bands to disappear). I now use 0.05% Tween or no Tween at all if it will be stored for a couple of days (over the weekend blocking, etc...). Lastly someone mentioned using a marker to mark the molecular weight bands before blotting. Great idea and I do it myself. Takes the worry out of what happens to the markers all together. The only problem I have with markers are the See-Blue which have one red band and all the rest are blue. The red band always disappears. I have never given it much thought since it is marked by me before continuing to the next step. Just be sure the blot does not dry out while marking the bands. You will need to blot off the area where the markers are so the pen does not spread out, but do not blot the rest of the membrane. Leave it wet.


Yes, but be careful. Losing markers from your blot may indicate protein leaching. An ink mark may tell you where your marker was, but wonīt help if your target protein ha also gone!

#9 mikew

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Posted 13 March 2009 - 01:30 PM

Several things;

#1. Someone mentioned above about kaliedoscope markers losing color.
I have experienced the same on occasion. What marker are you using?
I have found the one from BioRAD to be the best (just stained blue).
#2. Do you know if you are really losing protein or merely losing the dye associated
with the ladder? This involves a positive control.
Do you do an actin probe or other antibody that you know works Western of this blot? Does it work?
I have lost the coloring of the dye on the ladder a couple of times previously with Invitrogen ladders.
The blots still worked fine. You may be losing dye, not protein. A positive control will sort this out.
#3. After your transfer, mark the position of the ladder bands with a pencil. This works fine and doesn't
interfere with the Western.

#10 Irene G

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Posted 16 March 2009 - 04:12 AM

Thank you everybody
We are going to make all the buffers new and we'll buy a simple non-fat milk without anything. We will see.

#11 Nrelo

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Posted 18 March 2009 - 02:51 AM

i used to block the membrane with 10% skim milk, but the background is quite high in ECL, so I have changed to 5%BSA. One key to reduce background is to block the membrane for longer period of time, say 2~3hr




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