<x-flowed>Hello,
I have a sample that is an inclusion body from brain tissue. The
inclusion body has been isolated from a histology slide by laser capture
technique. The inclusion body was initially visualized after staining
by PAS (after the usual fixation and mounting procedures used by
pathologists) and if you have very good eyesight individual inclusion
bodies can be seen with the unaided eye (not mine unfortunately).
The laser capture technique (as it was explained to me) works by
focusing laser energy on a selected part of the tissue with the aid of a
microscope. On top of the tissue is a clear disc with a polymer coating
on the contact surface with the tissue. The laser is aimed at the
appropriate spot and fired. This heats up only the polymer and causes
it to adhere to the tissue directly beneath where the laser is aimed.
One can remove the polymer from the plastic disc as a thin piece of what
looks like just clear plastic with only the targeted tissue adhered to
it.
Yes...... an investigator wants me to identify the major,
presumably glycoprotein, constituent from several of these inclusion
bodies. So far I have tried urea extraction, or guanidine extraction
followed by desalting on Zip tips or just dilution into matrix then
MALDI TOFMS analysis. I have also tried in situ CNBR digest in 70% TFA
for 3 hrs at RT followed by MALDI TOFMS and finally just boiling it in
SDS sample buffer and silver staining the gel.
The only result I have gotten is an ion signal at about 18,500 m/z on
the MALDI. The yield of this ion is so great that I think it is derived
from the polymer used to capture the inclusion bodies not to mention
that it isn't the molecular weight that is expected by the
investigator. Edman sequencing of the guanidine extract did not yield
any information.
Questions:
Has any one isolated protein from laser captured samples, or know of any
references that have done so?
Could the process of formaldehyde fixation, embedding and mounting on a
slide followed by PAS staining cross link the protein in the inclusion
body such that further analysis would be impossible?
This is a dense stained inclusion body of about 0.1- .2 mm area (this is
a guess). Could there be enough protein to analyze and by what
technique?
Thanks in advance for any help and I apologize for the length of this
email.
Brian Hampton
Project Leader
Protein and DNA Sequencing Core Facility
Hematopoiesis Department
American Red Cross Holland Laboratory
15601 Crabbs Branch Way
Rockville, MD 20855
301-738-0814
hampton@usa.redcross.org
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