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Western unresolved lanes


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#1 fedemarz

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Posted 31 December 2010 - 08:51 PM

Hello, Im Federico from Argentina. This is my first time here.
I have this problem: Something is happening in the electrotransference.It seems that the lanes come united. Cuold anyone help me with this? 3 months with this problem !!

Conditions:
SDS-PAGE 25 ul Proteins, 30mA, 1hs
ELECTROTRANFERENCE: 4hs at 250mV, electrotranference solution 1X methanol 20%. BioRad Mini Protean 3 Western transblot.
I send a photo..
Thans for your time.

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#2 dcch

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Posted 01 January 2011 - 03:42 AM

Happy new year.

I have used this system before (now shifted to semi-dry) without any problem. Apparently, your Ponceau S membrane staining result showed good protein transfer. Just two cents of my view:

1. When you casted your SDS-PAG, make sure you did not overlayed the seperating portion with water. Isopropanol or butanol will do this job better. We discovered that sometimes the protein may leak and cross well when it is moving from stacking to seperating region. Did you observe similar case if you stain your gel with commasie blue? This is important to distinguish whether the problem occurs in gel itself, or during transfer.

2. After you assembled the sandwich, make sure that you press and roll the sandwich again and again (with falcon tube, etc). This is to prevent bubble and ensure total contact between gel and membrane.

3. Are you running the system in cold room or surrounded by ice bath? In our lab we think that the icepack given alone is not enough to maintain the cold temperature. We used cold transfer buffer with ice surrounding the tank. Maybe you can try in cold room?

4. Lastly, use freshly prepared tranfer buffer. Methanol may evaporate away very fast if you store it in room temp. Otherwise, try to store it in 4oC.

Edited by dcch, 01 January 2011 - 03:44 AM.


#3 fedemarz

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Posted 01 January 2011 - 02:08 PM

Happy new year !! Thanks for your time dcch !!

Ive already stained the gel with coomasie blue and it seems that theres no problem..I obteined resolved lanes..For that reason I thought tha the problem is in the electrotranference.

I didnt try working in a cold room...I replace the ice periodically, maybe it isnt sufficient...I remember that the tank is quite hot when replace the ice. Peharps the problem is there...

In january I will return to the lab I hope that the ice is the solution..
Thanks foy your help !!




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