I am trying to perform a western blot against a protein expressed in plants. The antibody is well-characterized, the protein target is abundant, and I have previously visualized the protein in western blots using chemiluminescent development methods. But I wanted to use a Li-COR Odyssey instrument, with fluorescent secondary antibodies, because it is more quantitative than chemiluminescence. To this end, I homogenized plant tissue in a strong lysis buffer (TBS with 1% NP-40 and 10% glycerol + protease inhibitors), added Laemmli buffer to the soluble fraction, boiled this, separated proteins by SDS-PAGE, transferred to nitrocellulose, probed the membrane with the same primary as before, and then used the IR680 secondary prior to scanning on a Li-COR Odyssey scanner. Sadly, the result was disappointing. I observed detection of many plant proteins. I am worried this is due to autofluorescence. Soluble plant protein extracts tend to be very green in color, indicative of light-harvesting pigments that absorb in the red and far red. I am concerned that the Li-COR methodology may not be applicable to soluble protein extractions from plants unless they have been completely depleted of pigments. Is this true? Does anyone else have experience with using a Li-COR to visualize proteins from plant extracts?
Just to double check, are you using the Oddssey nitrocellulose membranes?
Reason I ask is the Licor scanner needs special type of membranes to limit the background from traditional PVDF and nitro membranes.
I use nitrocellulose from biorad, but so do others in my lab who do not work with plants, and they do not observe the background I am seeing.
Is their nitro membrane compatible with infrared scanners?
Can you upload a picture.
Check this site out
https://www.licor.com/bio/products/reagents/membranes/
Hi Damian,
There are some papers (I guess in the Li-Cor web site) which indicates all the specification to reduce the background. At the begining I had a lot of troubles with that, but finally I got really god blots. I never use the Li-cor membranes (Amersham or Biorad only). I think that the most important point was no detergents in the last washes. Always fresh transference and PBS buffers.
Now I remember that the red (800) secondary antibody had more autofluorescence, usually I used the green one.
Never stain with Ponceau when use Oddyssey (really enhance the fluorescence).
May be you need to change or modified the extraction buffer, although I used a similar one (PBS, instead TBS and SDS instead of NP40; and also glyserol.
You can also try loading less amount of protein and try all the Oddyssey recommendations (attached too)
And always scan the membrane BEFORE incubate with any antibody (because unspecific fluorescence could be due to unspecific binding of antibodies).
I attached the paper that I said (but I've never had to try trichloroacetic- precipitation, I got the success before try that!)
If you need I can send you all the specific buffers and procedures that I used (I attached one of our group publication with some detail of the western blot protocol that we use, but you can ask for more detail).
Hi Damian,
I am having what seems like the same problem as you. I have been trying to trouble shoot this for about 6 months now, attempting to optimize my blotting protocol, dilutions, buffers, timing, membranes, antibodies, etc. Nothing seems to work. I have been in contact with licor tech support and they really were not much help except that they said chlorophyll autofloresces at 700nm (duh..) and certain plant proteins will too. I am convinced the licor is simply not meant for plant samples so I will be converting back to chemiluminescence once I find or make an appropriate dark room. Note that I am working with total membrane protein extracts from Arabidopsis thaliana and chlorophyll has been removed with repeated chloroform methanol precipitations. I have attached an image of an electroblot on nitrocellulose that has not been blocked or treated with any antibody, the amount of autoflorescence is incredible. I have also attached an image of the same electroblot blocked and treated with primary and secondary antibody. Can you tell the difference?
Best,
John
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