 |
 |
Dear Martin, Thank you for sharing your observations with me. I believe your hypothesis makes a lot of sense. I think we can give it a try with longer and stronger glow discharge settings or we stick to carbon-copper grids on this protein complex. I wish you all the best. Yours sincerely, David On Fri, Nov 17, 2023 at 6:42 PM Martin Høgholm Jørgensen <[log in to unmask]> wrote: > Hi David, > I have experienced this with two protein complexes I have worked with. The > first case was an extremely high-affinity complex that was nonetheless > dissociating on grids. This gave rise to such small (and probably partially > denatured) particles that the images looked empty, but the different noise > distribution convinced us that it was actually overcrowded by these > low-contrast, residual particles. Diluting the sample to extremely low > concentration (similar to that suitable for neg stain) showed that this was > indeed the case. This happened on any grid type I could get my hands on and > in all the buffer and additive combinations I tested. Finally, adding a > high-affinity binder rectified the behaviour on carbon-copper grids but not > on gold-gold. My best hypothesis to explain this was that the dissociated > subunits maybe bound the carbon foil preferentially compared to the AWI > while this was not the case for the gold foil (it is my impression that > this is the case for most samples as images of gold foil usually show a > very sparse distribution of particles while carbon foil is completely > covered). I imagine that adding the binder increased the stability of the > complexes just enough that they would outcompete the dissociated subunits > for binding to the AWI on carbon grids but not on gold. > > The second sample I worked on shortly after, and there I saw the same > overcrowding of small particles on gold grids. Based on the experience from > the above example, I tested the sample on carbon grids instead and I got > intact complexes without any modification to the sample. > > If the hypothesis holds true, it is possible that you could reach the same > results with gold as for carbon by glow-discharging the gold grid for > longer or with a higher current, but I never went on to test this. > > I hope this might be of some help or at least interest. > > Sincerely, Martin H. Jørgensen > > ------------------------------ > *Fra:* Collaborative Computational Project in Electron cryo-Microscopy < > [log in to unmask]> på vegne af CF David Hou <[log in to unmask]> > *Sendt:* fredag, november 17, 2023 10:55:49 PM > *Til:* [log in to unmask] <[log in to unmask]> > *Emne:* [ccpem] Protein particles disappear on gold grid (AltraAuFoil) > > Dear CCPEM community, > > Have you ever encountered that your protein sample can be seen on > copper-carbon grids but when doing the same condition on the gold-gold > grid, there are no particles. > > The protein sample is an oligomer to an approximate size of 200 kDa plus > pre-incubated with short DNA fragments (~40 bp). Protein complex pI:5.3 in > pH 8 buffer, and DNA to protein complex ratio is 1.5:1 > > We would like to improve our data quality so that the protein can be > collected on the gold grid. Any input would be appreciated. > > Yours sincerely, > > CF David Hou > > ------------------------------ > > To unsubscribe from the CCPEM list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1 > > ######################################################################## To unsubscribe from the CCPEM list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1 This message was issued to members of www.jiscmail.ac.uk/CCPEM, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/