Hi, I hope you are doing well,
I am working to remove DNA from protein sample by using silica hydroxode magnetic beads. I used different binding buffers but I din't get my protein band in initial supernatant. I don't know where my protein goes, maybe it is attached to magnetic beads along with DNA. I tried different solution to make sure that my protein not attched to beads but unfortunately I did'nt succeed.
I need your assistance in this matter that what should i add with my binding solution to avoid protein binding with beads and I can easily get in my initial supernatant.
I will be very thankful for your kind suggestions.
Binding Solution: (Tris-HCl + NaCl + 1% SDS + 40% Isopropanol) sometimes I use PEG of 5-30%.
Are you sure your protein didn't precipitate due to the high amount of isopropanol? For keeping proteins in solution: Organic solvents = bad, detergents = good, urea / guanidine chloride = very good. May affect DNA binding to silica.
If unsure if your Houdini proteins just clinged to your beads, harvest the beads and boil them in SDS-PAGE loading buffer.
Andre Marschall The isopropanol (40%) is optimized in my protocol because I tried different percentages of Isopropanol and I only got bands on 40%, but there is something missing in my binding buffer, due to which I can't get my protein in supernatant. Yesterday I tried 6M and 8M urea along with my binding solution and I got light bands for protein in SDS PAGE, but now the problem is that I also got bands in Gel electrophoresis in supernatant sample, which I don't need. I just need protein in SDS PAGE and want to attach the DNA to magnetic beads and then elute it throgh elution buffer in final step.
if you have idea to increase protein band in SDS PAGE and avoid DNA band in Gel electrophoresis, please tell me.
Thank you for your kind response.
If you want to bind DNA to silica, it likes a pH below 5. Typically it's around pH4, in the presence of high concentrations of chaotropic salt (eg. guanidine chloride or thiocyanite). This is how any silica DNA prep protocol works. For which Tris is the wrong choice, usually it's an acetic acid/acetate buffer. Did you retrieve any DNA thus far?
Typical recipes for DNA prepping precipitate the proteins in the presence of SDS and followup KCl, resulting in potassium-SDS coprecipitation with all the cell debris and such. If you require genomic DNA, avoid the basic lysis step, as it will result in a wild entanglement of DNA that precipitates as well. Smaller DNA pieces are soluble though.
There is also the phenol-chloroform-extraction of DNA. The DNA will precipitate from the biphasic system, the protein will locate at the interface. You could retrieve said protein interphase in SDS-PAGE buffer for analysis, removing the excess solvents afterwards.
You can mix and match appropriately. Just mind that guanidine chloride doesn't mix well with SDS and forms a precipitate, you'll have to dialyze these samples (droplet dialyzation on a buffer swimming hydrophilic membrane filter works fine for the purpose of mere analysis).
Andre Marschall Thank you for your detailed explanation. I will change my protocol and will see if it works. I will also try low pH and high concentrations of Chaotrophic salt.
About phenol-chloroform, we don't use that, we just focused on using magnetic beads to check whether it works or not.
Thank you
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