[3dem] Leica EM GP2 best practices

[Guenter Resch]

Hi Andrey,

I have been a part of the development of both the GP and well as the GP2, so I am happy to help here ...

> 1.       Do you use the blotting sensor? In my case it fails very often and
> it seems that the results are better without it. 

I absolutely recommend to use the sensor (also because of your description in question 4), so I wonder what kind of failure you are observing? Is the blotting arm moving too far or too little? What amount of liquid are you using, is that liquid an aqueous, clear solution? The sensor should work well for > 3 ul, if that is not the case, a recalibration might be needed.

> 2.       In case you use the blotting sensor, what range of "additional
> movement" do you use?

For each sample and each sample volume, I am optimising this distance so that the grid is slightly bent. (This distance is the forward movement between moment the filter paper gets wet and the final blotting position, so it depends on the size of the droplet, if the droplet is spreading on one or both sides of the grid, etc.)

> 3.       In case you use manual blotting, how do you set the horizontal blot
> position: just by touching the grid with the filter paper, or slightly
> bending it?

See question 2.

> 5.       What range of blotting time and specimen volume do you typically
> use for SPA samples at conditions specified?

There are a few examples (and also my "default protocol") in our paper from 2011 in CSH Protocols: https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ncbi.nlm.nih.gov_pubmed_21724813&d=DwICAg&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=vwwoS1Ro0hBrIltxDmLtX8JlWGmeJyTGZlRgV0xlDEFpSF0vtBOSbBRvt6WnUVka&s=ijWUyPYwu4PORRp6G9ZrsBKv9AkI-tfkv5bv9LKbjcI&e=  . Of course, these were tested to the "original" EM GP, but as the operating principle has not changed, these recommendations should still apply ...

Good results can also be obtained with completely different combinations of parameters (e.g., longer but weaker blotting), so these recommendations are only one point in the multidimensional space of parameters that works quite well.

Best regards and have a nice weekend,

	Guenter

-- 
Dr. Guenter Resch
Nexperion e.U. - Solutions for Electron Microscopy - https://urldefense.proofpoint.com/v2/url?u=http-3A__www.nexperion.net&d=DwICAg&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=vwwoS1Ro0hBrIltxDmLtX8JlWGmeJyTGZlRgV0xlDEFpSF0vtBOSbBRvt6WnUVka&s=Q4JjbDYms69ea4zNirov9z-DdvXLO2d9cF-fPyQ9SUM&e= 
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