Hello,
I currently have my T4 virus in HEPES buffer. Unfortunately, there are always crystals that are contaminating my solution (due to the buffer in which the Viruses are) when I observe them under an electron microscope. I tried centrifuging (15 min at 3000 g than 10 min under 5000 g) but the difference was very minimal. Prior to observation (without Negative Stain), I mix them with either PA7 or PAG gold beads (for electron holography purposes).
I am looking for buffer suggestion mainly that is compatible with all that and that is stable (under 4°C).
Elio Karim
PHD Student, CEMES-CNRS & CBI
CEMES-CNRS, I3EM Team 29 rue Jeanne Marvig
B.P. 94347 F-31055 Toulouse Cedex Office A200 CBI-LBME, Gleizes Team Paul Sabatier University 118 Route de Narbonne 31062 TOULOUSE Cedex Office 216
T4 should be pretty tolerant to most biological buffers so pick a buffer that you know is ok for your EM work and see if the T4 is stable in it. But I wonder whether the problem you are having is really from the HEPES or maybe from something else in the phage lysate. Did you try a control grid with only HEPES buffer to test?
Michael J. Benedik
Yes i tested the buffer on classic carbon grids (HEPES filtered One month ago) and i can always see the cristals which is wierd. I am trying to centrifuge at 8000g [2020, X. Jiao, Microbial Pathogenesis] to see what happens.
You could try either Tris or phosphate buffer to see if that helps. You don't need much buffer, 10mM or so should suffice to be sure the pH stays reasonably neutral.
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