Updated: October 11, 2002
N A N O P R O B E S E - N E W S
Vol. 3, No. 10 October 11, 2002
This monthly newsletter is to keep you informed about techniques to improve your immunogold labeling, highlight interesting articles and novel metal nanoparticle applications, and answer your questions. We hope you enjoy it and find it useful.
We apologize for the delay in sending this month's issue: this was due to changes in our e-mail server which resulted in a temporary problem with the list software. Please report any problems to webmaster@nanoprobes.com.
Have questions, or issues you would like to see addressed in the next issue? Let us know by e-mailing tech@nanoprobes.com.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nanogold-labeled Fab' fragments are the smallest commercially available gold probes that can be visualized in the transmission electron microscope. The unique surface coating of Nanogold means that it does not require additional macromolecule stabilization: because of this, and because the Fab' fragment is only one-third of the size of a whole IgG molecule, Nanogold-Fab' is much smaller than other immunogold probes, and can penetrate up to 40 micrometers into cells and tissues, label targets which may be inaccessible to larger colloidal gold probes, and label much more densely than colloidal gold.
Guo and co-workers demonstrate these advantages in their studies to localize secretory carrier membrane proteins (SCAMPs) in mast cells, and study their role in regulating exocytosis. Anti-SCAMP and secondary Nanogold-antibody conjugates were internalized by unfixed Streptolysin O - permeabilized mast cells, which were examined following fixation, silver intensification (47 min), embedding, and sectioning. The Nanogold labeling showed a previously unreported low level incidence of SCAMPS in the plasma membrane, where they colocalize and associate with exocytotic machinery near prospective fusion sites. Localization was confirmed using isolated mast cell granules labeled with 10 nm colloidal gold: these were washed by centrifugation in CB (137 mM NaCl, 2.7 mM KCl, 20 mM Pipes, 5.6 mM glucose, 1 mg/ml bovine serum albumin, pH 6.8), blocked, and labeled with monoclonal antibody 7C12 and goat anti-mouse IgG-10 nm gold (1:25). After fixing in buffered 3% glutaraldehyde and 1% OsO4, the granules were dehydrated in acetone, embedded, sectioned, and stained with uranyl acetate (50% methanol) and aqueous lead citrate. An oligopeptide, E peptide, unique to SCAMPs and highly conserved among SCAMP isoforms, was found to act later than other perturbants at a stage closely associated with membrane fusion: it is therefore a potential new reagent for analyzing the final stage of exocytosis, and these results imply a role for SCAMP2 in this process.
Reference:
Guo, Z.; Liu, L.; Cafiso, D., and Castle, D.: Perturbation of a Very Late Step of Regulated Exocytosis by a Secretory Carrier Membrane Protein (SCAMP2)-derived Peptide. J. Biol. Chem., 277, 35357-63 (2002).
Abstract (Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/277/38/35357
More information:
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nanogold® conjugates are ideal for pre-embedding immunoelectron microscopy, and many publications have described a wide variety or protocols for this application. Two of the most effective procedures, are reproduced on our website:
Recently, Murata and co-workers used Nanogold-labeled secondary antibodies with silver enhancement in a pre-embedding electron microscopy procedure in 6 micron cryosections to localize and infer the roles of the G1 and G2 isoform of Vacuolar H+-ATPase (V-ATPase) G subunit in mouse brains. G1 was distributed ubiquitously in the tissues examined, whereas G2 was specifically distributed in central nervous system neurons. Both G1 and G2 were strongly expressed in cortical and hippocampal neurons, cerebellar granule cells, and Purkinje cells. Electron microscopy and subcellular fractionation indicated that G2 was localized in synaptic vesicles, whereas G1 was not detectable. These results suggest that synaptic vesicles contain a unique V-ATPase with the G2 isoform, but not ubiquitous G1 subunit, and combined with enzyme activity data indicate that the enzyme with the G2 isoform is involved in synaptic vesicle acidification.
Reference:
Murata, Y.; Sun-Wada, G. H.; Yoshimizu, T.; Yamamoto, A.; Wada, Y., and Futai, M.: Differential localization of the vacuolar H+ pump with G subunit isoforms (G1 and G2) in mouse neurons. J. Biol. Chem., 277, 36296-303 (2002).
Abstract (Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/277/39/36296
More information:
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
We provide comprehensive technical support for our products on our web site, making the discussions as widely applicable and generally helpful as we can. Our technical support section support section includes an introduction to immunoelectron microscopy, including a discussion of the factors that can affect labeling performance of both primary and secondary antibodies, and how to test your labeling effectively before proceeding to a full TEM experiment, based on recent discussions on the MSA microscopy listserver.
Full details: www.nanoprobes.com/TechIEM.html
The MSA microscopy listserver provides a huge accumulated body of knowledge on immunogold and other labeling techniques, as well as other microscopy issues. Sign up, or search or browse the archives, at the MSA microscopy web site:
http://www.msa.microscopy.com/MicroscopyListserver/MLInstructions.html
A new reference book on immunogold-silver staining is now available, including three chapters written by Nanoprobes scientists which include detailed, specialized descriptions of many of the methods and protocols used most effectively with Nanogold®, FluoroNanogold, and silver and gold autometallography. "Gold and Silver Staining: Techniques in Molecular Morphology" edited by Gerhard W. Hacker and Jiang Gu IS published by the CRC Press, Boca Raton, FL.
More information (CRC Press):
http://www.crcpress.com/shopping_cart/products/product_detail.asp?sku=1392&parent_id=&pc=/shopping_cart/search/search.asp!
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Albrecht and co-workers have been developing alternative approaches to multiple colloidal particle labeling, and describe some of them in their presentation at Microscopy & Microanalysis '02. Uniformly sized colloidal metal particles of differing elemental composition such as Au, Ag, Pd, Pt, Ru, Rh may be distinguished by Energy Filtering Electron Microscopy, EF-TEM; this relies on the electron energy loss spectra, EELS, of different elements to selectively image materials. Alternatively, particles of different shapes may be distinguished directly by TEM or SEM.
References:
- Albrecht, R. M., and Meyer, D. A: All that Glitters is not Gold: Approaches to Labeling for EM. Microsc. Microanal., 8, (Suppl. 2: Proceedings); Lyman, C. E.; Albrecht, R. M.; Carter, C. B.; Dravid, V. P.; Herman, B., and Schatten, H. (Eds.); Cambridge University Press, New York, NY, 2002, 194.
- Albrecht, R. M.: All That Glitters is not Gold: Approaches to Labeling for TEM. Microscopy Today, May/June 2002, p. 24.
At Nanoprobes, we have chemically functionalized clusters and nanoparticles prepared from other materials for use as labels for different applications. 2 nm platinum particles have been activated and cross-linked to antibody IgG molecules and Fab' fragments using the same methods used for Nanogold® conjugation; the conjugates were used to localize a pre-mRNA splicing factor in HeLa cells.
Reference:
Powell, R. D.; Halsey, C. M. R.; Liu, W; Joshi, V. N., and Hainfeld, J. F.; Giant Platinum Clusters: 2 nm Covalent Metal Cluster Labels. J. Struct. Biol., 127, 177-184 (1999).
Abstract (Journal of Structural Biology):
http://www.idealibrary.com/links/artid/jsbi.1999.4153/production
More information:
Covalently linked tetrairidium clusters have been used in the image analysis and structural characterization of viral capsids. Maleimido-tetrairidium was attached via maleimide linkage to the C-terminus of the hepatitis B virus (HBV) capsid protein. Although the clusters are not visible in unprocessed cryo-electron micrographs, they are distinctly visible in three-dimensional density maps calculated from them, even at only partial occupancy.
Reference:
Cheng, N.; Conway, J. F.; Watts, N. R.; Hainfeld, J. F.; Joshi, V.; Powell, R. D.; Stahl, S. J.; Wingfield, P. E., and Steven, A. C. Tetrairidium, a 4-atom Cluster, is Readily Visible as a Density Label in 3D Cryo-EM Maps of Proteins at 10 - 25 Resolution; J. Struct. Biol., 127, 169-176 (1999).
Abstract (Journal of Structural Biology):
http://www.idealibrary.com/links/artid/jsbi.1999.4120/production
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Further advances in methods for correlative fluorescent and gold localization using Nanogold® conjugates to target Green Fluorescent Protein were described at Microscopy & Microanalysis '02 by Joanne Buchanan and co-workers. Use of microwave processing greatly shortened all the steps.
Reference:
Buchanan, J.; Micheva, K. D., and Smith, S. J.; Microwave Processing and Pre-embedding Nanogold Immunolabeling for Electron Microscopy. Microsc. Microanal., 8, (Suppl. 2: Proceedings); Lyman, C. E.; Albrecht, R. M.; Carter, C. B.; Dravid, V. P.; Herman, B., and Schatten, H. (Eds.); Cambridge University Press, New York, NY, 2002, 160.
The advance publication by Stromer and co-workers mentioned last issue, describing double immunofluorescence labeling to identify colocalization of proteins in smooth muscles, followed by electron microscopy immunolabeling with protein-A - 12 nm gold to localize individual proteins to specific cellular components, has now appeared in hard copy.
Reference:
Stromer, M. H.; Mayes, M. S., and Bellin, R. .M.: Use of actin isoform-specific antibodies to probe the domain structure in three smooth muscles. Histochem. Cell Biol., 118, 291-299 (2002).
Abstract (courtesy of Histochemistry and Cell Biology):
http://link.springer.de/link/service/journals/00418/contents/02/00453/
Bunting and Selig report the electron microscopic localization of DNA in ultrascopic nuclear appendages of polymorphonuclear white blood cells, using in situ end-labeling with digoxigenin and labeling with ultrasmall gold-labeled anti-digoxigenin Fab fragments.
Reference:
Bunting, R. W., and Selig, M. K. Localization of DNA in Ultrascopic Nuclear Appendages of Polymorphonuclear White Blood Cells from Patients with Low Serum B12. J. Histochem. Cytochem., 50, 1381-1388 (2002).
Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/50/10/1381
Using Nanogold®, antibodies or antibody fragments for the detection of haptenated nucleic acid targets may be labeled site-selectively for greater retention of activity; Nanogold may also be conjugated directly to chemically derivatized oligonucleotides.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
To unsubscribe from this newsletter, simply reply to this message, and replace the subject line with "unsubscribe." Alternatively, visit our web site newsletter page at www.nanoprobes.com/Newsletter.html#unsubscribe. You will receive a confirmation via e-mail.
|
