Bone Proteins--Long Reply

From Todd: "Hi Donna. We would like to extract these proteins from whole
bone from rats.  Do you have any suggestions?  Thanks."

To Todd and interested others, this is a long reply. If not interested
delete now.

There are a couple of ways to look at or for angiogenic factors in bone.

Screening tests: Immunohistochemistry using monoclonal and polyclonal
antibodies on formalin-fixed, formic acid decalcified paraffin-embedded
sections. NeoMarkers ( <> ) has
several angiogenic antibodies which have been verified in paraffin embedded
tissue. As does Clontech, Zymed,  Upstate Biotech and BioGenex. Note: the
antibodies I have gotten from Santa Cruz work well for Westerns but have not
been particularly convincing in paraffin. You can then ascertain the
location of the factors; marrow vs. matrix, and thereby narrow down your
isolation techniques. Fluorescent secondary antibodies can boost the
detection of small copy number proteins. We have had good luck using
biotin-avidin signal detection with HRP or AP or both. Specifically we have
localized bFGF and VEGF in newly formed bone at fracture sites in rabbits,
rats and mice.

In situ PCR or RT-PCR: Since you will have sections of tissue for
immuno-examination, it follows you will be able to amplify the factors of
interest using PCR or RT-PCR techniques in situ if you have access to a
thermocycler. Deparaffinize the slides as usual to 70 % EtOH. Transfer to
distilled water for PCR or DEPC water for RT-PCR. We have not needed to
float our sections onto DEPC water. But we also are scrupulous about
emptying and cleaning our floatation baths every night. I can fax to
specific conditions to you upon request. Rule of thumb: If you have
literature information on reaction conditions and cycle times for solution
PCR you can use nearly the same cycling times for in situ (with an extended
annealing time on the end) and use fewer cycles. For example, if a solution
PCR protocol asks for 25 cycles, set up your in situ run for 8 - 12 cycles.
I like to incorporate dig-dUTP or biotin-dUTP into my reaction mixture in a
ratio of 1:10 with the other dNTP's. This incorporation facilitates the
localization of the in situ product in a straightforward manner with fewer
washing steps thus less likelihood of loosing your tissue. Molecular Probes
even has fluorescent-dUTP. I have not tried it.

Finally, Protein extraction followed by SDS-PAGE and western blotting.
Extracting protein from bone matrix can be accomplished on decalcified bone.
However, we typically do our protein analysis and western blotting on cells
we have grown in culture. They are purer and more numerous than those from
cadaveric specimens. Also, when we do use fresh tissue, it's typically the
marrow portion only. Titurated and filtered through a 70 um mesh to remove
bone spicules. Then centrifuged at approx 1000 rpm, to remove the fat, and
to loosely pellet the cellular fraction. The cell pellet is resuspended in
RIPA buffer (I can fax our receipe or you may reference Molecular Cloning or
Current Protocols in Molecular Biology) titurated sequentially with a 5 ml
pipet then a 2 ml pipet). Centrifuge at 4 oC save the pellet for membrane
bound protein extraction and use the supernate for soluble protein
fractions. Using the Bradford method or one similar, obtain the protein
concentration of the supernatant. Load 100 ug of protein per gel well and
run the gel at constant voltage till the gel front is 1 - 2 cm from the
bottom of the gel. Transfer overnight at constant amperage onto a prepared
nitrocellulose membrane. Rinse thoroughly, place in a hybidization bag with
desired antibody and incubate at 4 oC overnight with rocking. Develop the
antibody reaction as desired the next day. Again, I'll fax you the specifics
on washes and times.

As you can see, If your wanting to localize the protein do IHC. It faster
cheaper and usually adequate. In situ PCR techniques can assist in detection
of low copy number proteins as can fluorescent secondaries. SDS-PAGE and
Western blotting is often seen as the definitive method for showing
differences between treatment groups in tox studies or for gene expression
analysis. But it is time consuming and problematic. I've been asked to
"write this up" for the JOH. I will try to do so. Good luck, Donna

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