Hi, does anyone have an idea what might be causing this dark brown (pale, clear yellow if I re-suspend) precipitate to form? Here is some background on my sample preparation:
1. Lyse HEK293 cells with RIPA buffer (inhibitors added include PMSF, DTT, Leupeptin, Aprotinin, and Pepstatin). The extracts are clear at this point.
2. Pull-down biotinylated proteins using magnetic Thermo Fisher Scientific Dynabeads MyOne Streptavidin C1 (beads conjugated to streptavidin). These beads form a murky brown slurry and I suspect SOMETHING from these beads might be creating the precipitate later on.
3. Wash the bound beads first with a stringent buffer with a high concentration of SDS, then a few washes in RIPA lysis buffer (more mild), followed by TAP lysis buffer (even more mild), and then 50 mM ammonium bicarbonate to finish.
4. On-bead trypsin digest to get peptides. To do this, I first reduce the disulfide bonds using DTT and then alkylate using iodoacetamide. Next, I do the on-bead digest and then collect the beads by centrifugation and using a powerful magnet. The solution I'm left with is clear at this point.
5. After speed vacuuming my solution for a few hours, my entire sample is concentrated and turns a dark, rusty brown colour. This precipitate easily disperses, with some pipetting, in 0.1% TFA and tinges my entire solution a translucent, faint yellow.
Anyone have any idea what is causing this? Just a side note that I do not use any strong acids throughout my protein preparation protocol.
Though it's not ideal, I can ZipTip the sample and the resin turns yellow when bound by my sample, however the resin turns white again after elution. I do not see the precipitate after ZipTipping but this could just be because my new, final sample volume is much smaller (~10 uL) after using a 10 uL ZipTip.
Dear Sanna Abbasi,
Intriguing question. If I understand your story correctly, there is a brown residue after the entire purification protocol with the magnetic beads?
If this is correct,my first question would be to learn from you if the beads you used were pristine, or already used in another protocol?
kind regards, Harrie Verhoeven
Dear Sanna,
Magnetic beads are not always as magnetic as you want them to be. We have experienced that in some high salt solutions the magnetism is less. This has impact on the pull down (less effective).
If your ZipTip has a small pore size, you should see a small layer of beads on top of the tip. That is what we see when we do the ZipTip (in-house produces ZipTips: http://pubs.acs.org/doi/abs/10.1021/pr900580n).
Does the yellow colour disturb your analysis?
Harrie, my beads are pristine. They have not been used in any other protocol prior.
Léon, the ZipTip resin did turn yellow after I pipetted my sample so it's possible. I have not analyzed the original yellow sample; after I ZipTip the sample, it turns clear, and the analysis of this clear sample seems fine.
Something else I'm thinking about; my beads are conjugated to streptavidin, which is a protein. Therefore, the streptavidin is probably also being digested by trypsin and released into my sample as well, so maybe this is responsible for the precipitate.
Sanna,
seems that you might have a starting point here.
Streptavidin itself might be digested, but I do not think it will contibute to the precipitate.
Dear Sanna Abbasi,
The answer by Leon may point in the right direction: the oxidation status of the iron (2+ vs 3+) may affect its magnetic properties. The 3+ state will be collected by the magnet, the 2+ state may escape. And after oxidation by oxygen from the atmosphere, it will form a brown precipitate. As for the theoretical background:
http://chemed.chem.purdue.edu/demos/main_pages/6.3.html.
I hope this is helpful. Kind regards, Harrie Verhoeven.
Dynabeads™ MyOne™ Streptavidin C1 - original website information
(https://www.thermofisher.com/order/catalog/product/65001#/65001)
"Are Dynabeads magnetic beads compatible with dithionite, DTT, and EDTA?Answer
No. Not only is dithionite a reducing agent, but the strong affinity of the dithionite ion for bivalent and trivalent metal cations (M2+, M3+) allows it to enhance the solubility of iron, making it a chelating agent. As a result, the iron in the Dynabeads magnetic beads is reduced and pulled out when they are exposed to dithionite. The same is observed if Dynabeads magnetic beads are exposed to DTT and EDTA."
More about the nature of the iron in the beads:
Dynabeads® magnetic beads are uniform, non-porous, superparamagnetic, monodispersed and highly cross-linked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The magnetic material within the Dynabeads® magnetic beads consists of a mixture of maghemite (gamma-Fe2O3) and magnetite (Fe3O4).
https://www.thermofisher.com/de/de/home/technical-resources/technical-reference-library/nucleic-acid-purification-analysis-support-center/dynabeads-nucleic-acid-purification-support/dynabeads-nucleic-acid-purification-support-getting-started.html
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