I Have a Crystal in my drop. my I don't know really what is it, a protein or salt or contaminant ! I want to apply SDS PAGE to understand it. But what is the best way to handling the crystal and best buffer for washing it?
For sure! I strongly recommend shoot it and collect the data first. If it is a salt crystal you will recognize it as well during X-ray data collection (few very strong spots at high resolution).
If you still want to put it on an SDS-PAGE: I would wash it in your reservoir solution and then directly put it into a few ul of 1xSDS sample buffer. Heat it, run all of it on your gel and you will see if it was enough.
You can still do that AFTER X-ray data set collection!
Good luck.
cheers Sven
I guess you use vapour diffusion for crystallization.Use your well solution to wash your crystal(some amount of protein is still in the drop), after you need to dissolve crystal in proper buffer.Try to use different pH then you have in crystallization solution. Difficult to say in advance. Better do this experiment if you have not only single crystal. In this case check X-ray diffraction!
For sure! I strongly recommend shoot it and collect the data first. If it is a salt crystal you will recognize it as well during X-ray data collection (few very strong spots at high resolution).
If you still want to put it on an SDS-PAGE: I would wash it in your reservoir solution and then directly put it into a few ul of 1xSDS sample buffer. Heat it, run all of it on your gel and you will see if it was enough.
You can still do that AFTER X-ray data set collection!
Good luck.
cheers Sven
Hi, I agree with both Valeriya and Sven about shoot first and ask questions later. Depending upon the objective, whether you want to just confirm that it is your protein, or a more sophisticated question like is it a modified form that crystallized or are their other molecules that trail along with it to allow crystallization how you do the washing could be important. To wash, we typically make several small drops of mother liqueur and then wand (using the crystal mounting loop) the crystal through them to reduce any of the diffusable or non-crystallized proteins. Sometimes, however, the crystals fracture and dissolve instantly. In that case, use mother liqueur with a slightly higher concentration precipitant. Alternatively, if you have a large number of very small crystals you can harvest the drop, spin them down in a microfuge and was the pellets. This works well. On the other hand, if you want to both confirm its protein and determine whether its modified or has a bound ligand you can use something like a zip-tip (C18 or similar resin in a pipett tip) to desalt your crystal preparing it for SDS-PAGE and Mass spec. There are a large number of variations of these approaches and you will need to adapt them to your specific needs.
I think you can realize very easily what kind of crystal it is by manipulating it using a very fine plastic fiber. A salt crystal will be very strong and won't show any cracks. A protein crystal will be very fragile. Then according to previous answers, XRD and then SDS PAGE or even more precise and easier in preparation size exclusion HPLC
If can shoot it real quick that might give you that best answer in the shortest amount of time, but other steps to consider are as follows (some of which you may have already completed):
I would do an optimization tray with small incremental adjustments of conditions, e.g. changes in pH and concentration of salt and precipitant. Included in that would be exact repeat of crystalizing conditions, maybe in triplicate, to hopefully obtain more crystals. If you have not already make sure you run negative controls for each condition, i.e. protein diluted in the buffer condition as well as buffer (-) protein from the last purification step (if you get a crystal with this negative control you know if is salt crystalizing).Make sure you note crystallization period. If it happens within a day or two it is probably salt crystalizing.
Eventually if you want to run SDS-PAGE, you carefully take the crystal and place it in an epi tube containing the same crystallization medium (and temperature), gently centrigure, remove sup, repeat 3-4x. Finally dissolve crystal in SDS-PAGE loading buffer and heat at 95C for 5 mins, etc as you would for running western or coomasie analysis.
No need to perform SDS-PAGE. Just add protein specific dye
Ref: Identifying, studying and making good use of macromolecular crystals(Acta Cryst. (2014). F70, 993–1008)
When I have one single crystal the first question I try to answer is: Can I get a structure from that single crystal? On the way of figure this out you will find many answers. I would shoot it first, no doubt. And off course in parallel try to reproduce the crystalls.
Careful because if you run the crystall after shooting on a SDS PAGE the protein might be hardly broken.
If you still want to run it on SDS PAGE I do like Andrew.
Good luck!
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