Hi Xiao, I would poke the crystal with a piece of glass fiber or an oocyte needle to make sure it is not salt. Some protein crystals can be a little tough (rubber-like), but they react to poking very differently from rocks (salts) and will eventually shatter. So far I have found this test to be the most reliable before an X-ray diffraction image. You can also try IsIt (0.1% methylene blue) staining, glutaraldehyde crosslinking/staining (causing protein crystals to become yellow due to Schiff base formation) and so on. If you can see a band on SDS-PAGE gel and still want to confirm that it is your protein, you may consider MALDI-MS which is very sensitive, maybe too sensitive (bear in mind that due to potentially insufficient wash, the protein band or MALDI peaks may come from the solution not the crystal).
Nevertheless, shoot the crystal! Zhijie From: Xiao Xiao Sent: Thursday, September 25, 2014 7:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off-topic;Crystal cannot dissolved in buffer 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?
