I am using Seprion PAD-beads to isolate my target and I need to do immunogold labeling Electron Microscopy (IL-EM) on my final sample. The elution method suggested by the kit is the following:
1.Add 10-50 μl of 0.1 M NaOH, 0.1% Triton X-100, resuspend the beads and
shake for 5 min.
2.Place the tubes on a magnet to capture the beads.
3.Still on the magnet, add the same volume of 0.1M HCl to neutralise the alkali and mix briefly by pipetting up and down.
Unfortunately, using this protocol I generate NaCl which interfere with the following technique (EM). Do you have any experience/suggestion that could help me?
I think I should use a high pH solution and then neutralise it, but I do not know which system can give me a "salt crystal-free" sample.
Many thanks,
Rocco
Dear Rocco,
The 0.1M HCl is a way to neutralise the eluted solution. You might want to use another low pH buffer like citrate buffer or acetate buffer. First you will have to find out the ratios. This is quite easy to do: take 1 ml elution buffer and add the low pH buffer until the pH meter shows ~pH 7.5
The volume of the eluate may now be too large. Also the other salts that have formed no may interfere with the EM-staining.
My suggestion would be to keep your original elution protocol including the HCl. The eluate must then be cleaned from the salts. There are several ways to do that: dialysis, size exclusion chromatography or with concentrator spin-tubes. I would prefer size exclusion or de-salting columns. This is a very fast way to achieve the goal. With the concentrator spin-tubes you have the possibility to concentrate and buffer exchange in one or two steps.
Good luck
http://info.gbiosciences.com/blog/bid/197554/Biological-Buffers-pH-Range-and-How-to-Prepare-Them
Dear Duco,
I agree with you, salts will always form so it is better to get rid of them after elution. I will probably go for desalting columns, which seems to be very quick and easy.
Many thanks for your suggestions,
Rocco
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