Has anyone used these two products together successfully? I was told I need to use a low fluorescent (LF) membrane when trying to visualize the proteins after transfer from a stain-free gel (since my normal Immobilon-P membrane gave lots of background fluorescence), but I don't want to use Biorad's LF membrane because it's very expensive and more than double the price of Merck's LF membrane. I'll be using the FastCast stain-free gels from Biorad. Any advice is much appreciated!
I never used the Biorad's stain free gels, but I make my own gel according to the paper published by Ladner et al. on which the Biorad's gel is absed on (TCE, trichloroethanol) and used the Merck's LF membrane without any problem.
I enclosed the Ref. for the paper.
Visible fluorescent detection of proteins in polyacrylamide
gels without staining
Carol L. Ladner, Jing Yang, Raymond J. Turner, and Robert A. Edwards*
Analytical Biochemistry 326 (2004) 13–20
Janos
Nitrocellulose has very low background autofluorescence compared to PVDF, though LICOR shows Imobilon FL works well too. In my initial tests, the TCE laden pre-cast gels (Bio-rad TGX) transfer less effectively as more UV is applied to visualized the gel. Presumably the crosslinking is increasing protein/gel mobility. The gel imaging was very handy to see this effect. I recommend keeping UV exposure time to a minimum when visualizing this type of gel if the bands of interest are of low concentration.
my own experience Nitrocellulose works better than PVDF in this case. we dont have biorad chemidoc so i expose my runned gel under uv (not an auto option on our imager) then when i compare uv pic of total protein to chemiluminescence signal it is still too low( quantitative response is low but the graph is still linear). even when i increased the activation period of gel to more than 5min. any idea what could be the issue here? literally gapdh is working better than total protein in my case. so weird...
Hi Sepideh, maybe increase the concentration of TCE? When I hand-cast TCE gels, I use a final concentration of 0.5% TCE.
thanks Tanja. i have purchased a kit ready to use: http://www.bio-rad.com/en-ca/product/polyacrylamide-gel-reagents/tgx-stain-free-fastcast-acrylamide-solutions
dont know why it doesn't work well...
Since the initial post of this thread I have purchased the Immobilon FL membrane and in my hands is much better than Amersham Protran 0.2 nitrocellulose with respect to autofluorescence background signal using a Licor Odyssey. Very impressive. I've also used Bio-Rad Criterion precast gels with this membrane successfully using the wet transfer method at 20V overnight (< 100mA).
Has someone already used Stain-free as loading control and published in paper?
A simple search in pubmed will bring this up. See Guiterrez AND stainfree AND Ponceau.
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