By Renato V. Iozzo
Renato Iozzo and an authoritative crew of investigators current for the 1st time a accomplished and updated selection of effectively reproducible preparative and analytical equipment for the in-depth research of those very important compounds. that includes step by step protocols, this publication will allow either beginner and skilled researchers to isolate intact proteoglycans from tissues and cultured cells, to set up the composition in their carbohydrate moieties, and to generate options for prokaryotic and eukaryotic expression. There also are distinct recommendations for the suppression of particular proteoglycan genes, for the detection of mutant cells and their degradation items, and for learning particular interactions among proteoglycans and extracellular matrix proteins and with progress elements and their receptors.
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Additional resources for Proteoglycan Protocols (Methods in Molecular Biology)
Notes 1. Fresh formamide should be used and, once opened, the bottle stored tightly capped at –20ºC until further use. 2. Omnifit glass columns were adapted for HPLC with Tefzel tubing (1/16-in. 01 in. internal diameter) by boring out the ferrules with a 1/16-in drill bit (6). 3. Stains-All, or 1-ethyl-2-|3-(1-ethylnaphtho|1,2d|-thiazolin-2-ylidene)-2-, ethylpropenyl|naphtho|1, 2d|thiazolium bromide, is available from Eastman Kodak Co. with a catalog number of 2718. An alternative to Stains-All profiling of proteoglycans is Alcian blue staining (9).
Also, choose a 15-mL tube (for solutions up to 2 mL) so that during the neutralization step the H2 gas that is liberated is allowed to escape without the loss of the sample. 12. 1% casein in PBS is used as both the blocking agent and diluent, due to the fact that it gives superior backgrounds with the anti-HS antibody, 10E4. 13. If you plan to run gels with a separate stacker, do not remove the stacker before staining or blotting to nitrocellulose. 14. The SDS must be washed out of the gels with 3 changes in either H 2O or 50% ethanol prior to staining with Azure A, Alcian blue, or silver.
Solution 1. 30% acrylamide stock: 30 g acrylamide to a total volume of 100 mL H 2O. 2. Solution 2. 2 % bis-acrylamide stock: 2 g N, N-methylene-bis-acrylamide to a total volume of 100 mL H2O. 3. Solution 3. Tris-PO4 buffer (stacking buffer): a. 5 M). b. 25 mL 1 N H 3PO4. c. 8 with concentrated H3PO4. d. Make up to a total volume of 100 mL H2O. 4. Solution 4. Tris-HCl buffer (separating buffer): a. 5 M). b. 15 mL 2 N HCl. c. 8 with concentrated HCl. d. Make up to a total volume of 100 mL H2O. 5.
Proteoglycan Protocols (Methods in Molecular Biology) by Renato V. Iozzo