Hi, Thank you all for replying and giving me great suggestion in such a short time! This is a great community.
Here I try to summarize the replies I got, just in case if someone is interested: 1. Of course, the most straightforward and reliable way is to test it on the beam, period. 2. Since beam may not be available every time, another simple way is to use glass filer/metal needle/cat whiskers to poke crystal. Protein crystals usually easy to shatter, salt crystals are hard like rocks. And crosslinked crystal (especially happens when crystal is not fresh, or PEGs solution is old) is rubber-like, easy to bend but hard to break. Crosslinked will reduce resolution of diffraction. 3. Can tell whether it is protein or salt by IsIt (0.1% methylene blue) staining, glutaraldehyde crosslinking/staining (causing protein crystals to become yellow due to Schiff base formation). 4.Some people did mention, in a few cases, some protein crystals were hard to dissolve. 5. Using low pH(3-5), NaOH, or simply add SDS loading buffer and heat may help dissolve the crystal. Again, thanks a lot for all your help! Best regards, Xiao Xiao 2014-09-25 16:53 GMT-07:00 Xiao Xiao <[email protected]>: > Hi everyone, > > Sorry for an off-topic question. > > I got a problem with crystal dissolving. Basically I got crystals of my > protein in various conditions, most conditions contain PEGs but different > salts. These crystals has very similar shape, so it should not be salt. > > Now I am trying to dissolve the crystal to make sure it is my protein, by > SDS-PAGE and N-term sequencing. I washed the crystals in its original > crystallization buffer few times then transfered them into regular buffer > (500mM NaCl, 50mM HEPES 7.5) with or without 10mM DTT, however the crystal > didn't dissolve. > > I then tried to heat it then add SDS loading buffer to run a gel, I did > see very small amount of protein on the gel, at the correct position, but > it's not enough for N-term sequencing. > > Is it normal for a protein crystal? And does anyone have any suggestion > for dissolving such crystals? > >
