I plan to conjugate a protein with colloidal gold particles and, then, use in the transmission electron microscopy experiment.
Should I use a correlative concentration with biological activity (in this case, in cell culture)?
I will conjugate through a commercial kit, following the manufacturer's protocol. To correlate with biological activity, I would have to use the entire volume of the conjugation reaction. My concern is that they have many gold particles that compromise the ultramicrotomy.
Thank you.
Can you develop your experiment ?
Are you going to conjugate the gold to an antibody ? Or a protein that you'll later give to the living cells by uptake ? Or something else ?
Can you develop a bit more what you mean by "correlative concentration with biological activity" ?
Dear Nicolas,
I plan conjugate a recombinant protein and, then, treat tumor cells with this gold conjugate protein and evaluate internalization by TEM.
This recombinant protein has present cytotoxicity activity in tumors cells when we use 0.5uM.
Because each reaction of the commercial kit combines only 5 ug protein with gold particles, I would have to use all this reaction in a single treatment. So my concern is there an excess of gold particles compremeta the ultramicrotomy step. Thus, I thought of using lower concentrations, but not sure. I read some articles, but did not find details about this preparation.
Thank you.
Katia.
Hi !
1. Don't take it personally, but as a first advice, you could make your maths again. Just in case, since, I guess, these are not simple maths... ;-)
2. If you're maths were right, can you just try to downgrade your treatment to less cells, smaller well, smaller volume ? If you're treating animals and not cells in a petri dish, this advice of course does not apply...
3. Would it be possible to use only a fraction of conjugated proteins ? So you would have a mixture of 10% of tagged proteins and 90% of untagged proteins. Hence your preparation would allow you to carry out 10 experiments...
Hello!
Nicolas, don’t worry, I don't take it personally!!!
I contacted the manufacturer and they told me that I could conjugate 15ug, more than informed before. So, I carried out my experiment this week using smaller volume. As soon as I have results, I will tell you.
Thank you for your attention!!!
Kátia.
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