Hello,
For several months now, I am doing biotinylated proteins pull-down using MyOne™ Streptavidine C1 Dynabeads™. I cultivate the cells in 100mm dish, I wash them with PBS before I lyse them with RIPA buffer (50mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% Na deoxycholate, 0.05% NP-40, 0.5 mM EDTA, protease and phosphatase inhibitors). The samples are very viscous after lysis, therefore, I sonicate them using a bioruptor to degrade DNA (sonicator bath). Then, I loaded them on the beads (that were washed three times with RIPA) and incubate overnight with rotation at 4°C. The next day, I harvest the unbound fraction and I wash the beads several times before elution (not detailed here because not relevant). I did several optimisation steps with different ratios of beads and proteins. Every time, it worked fine.
Since few weeks, the bioruptor is broken. Since then, I did 3 pull-down experiments and only one worked : all the biotinylated proteins were found in the unbound fraction in the other two experiments, meaning that there was no binding to the beads (I am using the same batch of beads since the begining).
The only thing I changed was the breaking of DNA. For the first experiment, I sonicated the samples using an ultrasonic homogenizer (classical sonicator where a probe is put within the sample) and a foam appeared in the samples during sonication. For the second and third experiments, I used a 0.4mm needle and a seringe to break the DNA and a lot of bubbles also appeared in the samples (especially in third experiment).
I am thinking that the bubbles are impairing the binding of the biotinylated proteins but I don't understand how exactly. Also, I really would like to break the DNA, otherwise the samples are really viscous and hard to pipet, and they do not mix well with the beads.
Does anyone have any suggestion regarding this issue? Thank you!
It is possible that the SDS in your buffer is foaming and causing oxidation on your Cysteine Amino Acids.
The sonication of your sample was generated by heat, did you keep your sample on ice during this step?
Thank you for your reply Nicolas Poirier
I kept my samples on ice, but for sonication, which lastest max 10 seconds, I didn't have them on ice, but put them back right after.
And I am considering now an issue with the beads (maybe too old?) as I did another experiment today without sonication nor needle and it failed.
Justine Guguin you can try 2 x10 sec. Depending on samples and viscosity of the samples, cells can be sonicated again for a further 10 s .
https://www.assaygenie.com/sonication-protocol-for-cell-lysis?srsltid=AfmBOopXOwTbSwsNFY6XutR8Cn_lmKOIY2fwR5rgIsSCEXG0wkQAqfLv
Maybe the beads are too old? I can't say for sure, check by depositing your protein on SDS PAGE gel at each step.
I tried to change the beads today, but I did not have better results. Something that might be relevant and that I forgot to say before is that I was working mostly with 200 µg of proteins and 50 µL of beads. Recently (since the issue started), I increased the amount of proteins to 1 mg or more while still using 50 µL of beads. It worked only once. I am starting to wonder if the quantity of DNA present in my sample that make it very sticky can also be a cause of why it is not working, as if it might interfer with the binding of the proteins to the beads. However, I have one experiment for which I did not sonicate, nor use the needle and it worked, while I have one experiment for which I sonicated and centrifuged the samples and it did not work, so I am really confuse right now.
Yes try to go back to your original conditions (200 µg of proteins and 50 µL of beads).
Keep the same conditions by doing an absorbance assay (DNA) with a spectrophotometer or nanodrop. Dilute the samples if necessary in the buffer to have the same concentration by volume and therefore the same quantity of contaminants and salts because this can affect your binding of the biotinylated proteins.
Try different ratios, stirring, contact time to have a binding, and see if you get a better result.
Can you degrade the DNA by adding benzonase and magnesium chloride? 5 Units of benzonase together with about 10 mM MgCl2 works for us.
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