In principle this is straightforward, but you'll need reasonably-sized
crystals. You'll have to wash them very well in mother liquor (protein buffer
will probably dissolve the crystals). I do at least 3 serial transfers in large
(5-10 ul) drops (with a loop), and for each transfer I move the crystals
around with a clean loop for a few seconds to get rid of the crystallization
solution (containing soluble protein). Don't worry about crystals
cracking/breaking during washing. When the crystals are clean I just pipette
them up with some mother liquor and add SDS PAGE sample buffer and run them out
on gel as normal. You can run out the rest of the mother liquor in the last
wash drop as a control (to see if you got rid of all the soluble protein). If
the crystal habit is the same, combining crystals from slightly different
conditions should be ok.
Things get more complicated if you have small crystals or crystals embedded in
skin (protein in the skin is not necessarily the same as in the crystals).
Precipitation is mostly ok because you can get rid of it during the washing
step.
In terms of quantities, if you assume a protein concentration in the crystal of
~500 mg/ml, then a cube of 100 micron (= large crystal) contains ~0.5 microgram
(1 nl) protein. So if two of your crystal dimensions are very small (needles)
you'll end up with very little protein per crystal. Also, for each small
crystal you wash you'll introduce a relatively large amount of soluble protein
from the drop. If you have needle clusters it could work, if you can
transfer/wash the entire cluster.
HTH, Bert
________________________________
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of sreetama das
[somon_...@yahoo.co.in]
Sent: Friday, May 02, 2014 9:58 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] verifying protein crystal content by mass spectrometry or
SDS-PAGE
Dear All,
Is there any protocol for preparing a sample from
(cleaning/dissolving) protein crystals for verifying their mass through mass
spectrometry or SDS-PAGE? How many protein crystals are required? Should they
all be from the same well, or can they be from different wells with slight
changes in protein concentration/precipitant concentration/ pH? Should the
crystals be cleaned with the reservoir solution and then dissolved in the
protein buffer?
I am getting very thin, needle-like crystals which are too tiny for mounting.
Adding Izit dye has also not been conclusive. Similar looking crystals have
appeared in 3 conditions. Changing precipitant concentration, protein
concentration, pH , and seeding have made no difference so far. I wish to
determine whether the full protein is crystallizing, or only some fragment. The
protein molecular weight is ~22KDa, and the crystals take ~3 weeks to grow.
Thanks in advance,
sreetama
