Hello,
I would like to ask your advice for a very specific question.
I need to use 1% Triton X-100 in PBS with inhibitors to extract proteins from tissue.
My goal is to analyze the Triton-insoluble pellet.
Here is my protocol:
1. Tissue: mouse brain hippocampus.
2. Homogenize with an electric handheld homogenizer.
3. Centrifuge at 13 000g for 10 min at 4C.
4. Separate Triton-soluble fraction (supernatant) and a triton-insoluble fraction (pellet).
5. Add 1x Laemmli buffer to pellet, analyze by SDS-PAGE.
The problem is that my pellet is full of genomic DNA which makes for very bad SDS-PAGE gels.
How do I remove this genomic DNA?
Could I spin the homogenate at 300g for some minutes and continue work on the supernatant (between step 2 and 3)?
Unfortunately I do not have access to a sonicator to shear the gDNA.
I have access to DNase1, but I'm worried it will not work because of my inhibitor cocktail.
Edit: I have added an image of the gel illustrating my gDNA problem. 1-4: insoluble fraction, 5-8 soluble fraction.
Blotting and antibody incubation confirms that my protein of interest is in the insoluble fraction, but of course the band is very messy.
If the protein(s) of your interest are reasonably stable in buffer/cocktail without the Triton X-100, proceed with conventional homogenization (not blending), followed by a 3000xg centrifugation to remove bulk of the nuclear fraction. This will eliminate most of the DNA (some nuclei will be damaged and cross-contamination is inevitable - mitochondrial DNA will also be there in the supernatant).
If the protein of your interest is in the nuclear fraction at this point, and it is "solubilized" by 1% Triton X-100, DNase may not work. However, you can take advantage of an anion exchanger to entirely eliminate the DNA and use what passes through. Of course, this may entail adding substantial amount of salt to ensure that majority of proteins (especially the ones that interest you) do not bind the matrix. This salt will have have to be removed or significantly reduced to have decent separation on gels.
Another crude but simple alternative is to add Protamine Sulfate, which will bind DNA and the complex can be precipitated in a centrifuge. This may not give you quantitative elimination of DNA but it may be helpful enough. There is size heterogeneity in protamine but it is small enough in size that some remaining amount in your protein fraction may not cause much interference. Of course, you will be optimizing the amount necessary to do what you wish to achieve.
Best,
Hi,
SDS-Page Clean-Up Kit could also be of help in removing the interfering contents.
Homogenisation of your TX100 insoluble pellet with syringe with narrow-gauge needle (10-20 passes ) may improve protein separation, even after you add sample buffer to your sample. If you have really small volume, you may try to re-suspend your pellet in RIPA buffer and see if you can extract more proteins and discard DNA pellet.
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