I have conjugated biotinylated antibody on a Miltenyi antibiotin antibody coated iron beads, which originally were meant to be used in cell separation. But I want to use it as a nanoparticle platform to deliever dual-antibody in animal model. How can I separated the beads from the solution then. If by centrifuge, what`s the centrifugal force and time I should set?
Because I`m new to nanotechnology, I wonder what`s the proper way to determine the concentration of the particles?
I havent tried to centrifuge, (I guess it should be possible) but these particles can always be separated from solution using a magnetic rack. If you add the particle suspension to a vial and then put the vial on a magnetic rack, particles will acumulate nex to the wall. You can easily remove the liquid, retire the magnetic field, and resuspend particles in a minimal volume of the desired diluent. Is the same effect of centrifugation at the end.
I havent tried to centrifuge, (I guess it should be possible) but these particles can always be separated from solution using a magnetic rack. If you add the particle suspension to a vial and then put the vial on a magnetic rack, particles will acumulate nex to the wall. You can easily remove the liquid, retire the magnetic field, and resuspend particles in a minimal volume of the desired diluent. Is the same effect of centrifugation at the end.
Hi, Gertrudis advice about separating iron NPs by magnets is sound. You really don't have to buy an expensive magnetic rack. You can do this using magnets from your local hardware store.
You can use light scattering to estimate the concentrations of the NPs, but determining the concentration of the NPs is not really important because you will have to use NPs as a negative control anyway.
What will be important for the in vivo is determining the Ab concentrations on those beads.
Thanks, Gertrudis and Steingrimur for your answer. Yesterday, I tried separate the NPS by a MS columns. I determine the antibodies coated on the iron NPS by substrating the amount of free antibody from the overall antibody before conjugation. But I still need a really direct evidence of successful conjugation of antibody on the iron NPs, so then I tried a SDS-PAGE gel electrophoresis of the conjugated iron NPs, hoping to find the band of 25KDa and 50KDa, but the result was negative. I wonder if the iron NPS in the sample will influence the electrophoresis process.
I have done IP studies using magnetic beads coated with antibody to immunoprecipitate cell components. Then I was able to do SDS-PAGE with bead suspensions and identify the band corresponding to the target antigen (in my case the antibody was a fragment covalently bound to the particles, so it mostly remained bound to the particles). So in principle it should be possible to detect the antibody. I used Dynabeads, not miltenyi beads, but I hope they should work as well. ? Take into account the expected amount of bound antibody and the SDS/PAGE sensitivity to design your experiment... Another alternative is an ELISA on magnetic particles, detecting bound antibody with a conjugate that onle reacts with your antibody and not with the antibiotin-coated particles. You could show and titrate binding in that way, washing the beads with solution using the magnet. betwen ELISA steps. The only problem is that this method is not truly quantitative (you have no standard).
Hi Chaoming. You are using biotinylated Ab. If the beads are streptavidin coated you could do a HABA assay to determine streptavidin occupancy.
Hi Chaoming, have you tried to use the BCA protein assay to quantitate protein on your beads?
Dear Gertudis Rojas, I tried running SDS-PAGE gel electrophoresis wil the antibody coated beads, but nothing came out. At the end of the procedure, I can see some brown colored bands on the loading area of the gel, which might be the iron beads I suppose, that can not pass through the 10% gel.
And also as Steingrimur Stefansson suggested, maybe I should tried BCA, but I think that the conjugated antibody should not be enough to calculate, I substrate the overall antibody I used by the unconjugated antibody of flow through, approximately 2.5ug antibody can be coated on every uL of microbeads, yet the concentration of the particles was unable to know.
So I would determine the conjugated antibody quantity in a indirect way that I mentioned. The next step will be to test the antigen combining ability of the antibody coated NPS by IF.
Yes, the brown material in the loading area corresponds to the beads, thats .normal Theoretically any protein that is non-covalently bound to the beads should go into the gel..Only proteins that are covalently bound and not released by breaking S-S should remain with the beads.
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