1 20 57
Fig. 3. Sulfate labeling experiment and GAG chain characterization. (A) Autoradiogra-phy of sulfate-labeled products of empty vector (lane 1) and aggrecan G3 construct (lane 2) from wild-type CHO cells (1). Note that only chondroitin/heparan sulfate GAGs are labeled (not the core protein). (B) Autoradiography of sulfate-labeled products of aggrecan G3 construct in Chondroitinase buffer (lane 1) and G3 digested by Chondroitinase ABC (lane 2). Note that the enzyme digestion almost completely eliminates the diffuse band.
2. Add 3 ^L of 20x Chondroitinase ABC digest buffer, 5 ^L of 10% Triton X-100, 5 ^L of 0.1 M NEM, and 2 ^l of H2O.
3. Add 15 ^L of Chondroitinase ABC solution (0.3 Units of Chondroitinase ABC).
5. Run samples in a SDS-PAGE gel, comparing to nondigested eluate (same as Subheading 3.4.). The Chondroitinase ABC-digested samples (sulfate labeled) are shown in Fig 3.
3.6. Detection of Proteoglycan Interaction with Heat-Shock Proteins Part I
1. (Steps 1-7 at 4°C) Combine spent medium (or cell lysate) with protein A beads and Hsp25 antibody, rotating for 5 h.
2. Spin at 14,000g for 15 s, then transfer supernatant to waste.
3. Add 1000 ^L of washing buffer D to each tube and rotate for 60 min.
4. Spin at 14,000g for 15 s, then transfer supernatant to waste.
5. Repeat steps 3 and 4 twice.
7. Carefully and completely remove all liquid from step 6.
8. (From here on, at RT) Add appropriate amount (e.g., 30 ^L) of 1x loading buffer with or without DTT.
10. Cool tube using running water.
12. Load supernatant onto SDS-PAGE.
13. Run the gel and process the gel as under Subheading 3.4.
3.7. Detection of Proteoglycan Interaction with Heat-Shock Proteins Part II
1. Transfect and label cells as under Subheading 2. (before sample collection).
2. (Steps 2-5 in cold room) Wash labeled cells (live on dish) twice with 5 mL of washing buffer E.
3. Lyse cells with 1 mL of digitonin lysis buffer (containing DTSSP), then scrape cells off dish.
4. Transfer lysate to a 1.5-mL Eppendorf tube and incubate on ice for 30 min.
5. Add 200 ^L of 10 mM glycine to sample (to deactivate excess cross-linker), then incubate on ice for 10 min.
6. Ni chromatography as under Subheading 3.3.
7. Electrophorese samples with dithiothreitol loading buffer (to release cross-linked proteins) as under Subheading 3.4.
1. All ingredients are sterilized, molecular biology grade.
2. A PCR hot-start program typically contains one denature cycle (94°C for 4 min, 72°C for 2 min and 55°C for 1 min) and 30 cycles of amplification (94°C for 45 s, 55°C for 45 s and 72°C for 3 min). Annealing temperature and elongation temperature need to be adjusted for different oligo lengths and templates. See a PCR publication for details.
3. Low speed spin for 15 s can be used to collect all liquid in the tube after inversion.
4. Stringent washes are necessary to eliminate background proteins and for clear visibility of the candidate proteins. Stringent washing buffer (wash 1) can be increased with higher molar concentrations of urea (utmost 8 M). If more stringency is needed, radioimmune precipitation (RIPA) components can be added to the buffer and subsequent washes. A repeat wash 3 is recommended if RIPA is added to wash 2 and wash 3, since deoxy cholate will allow different charges on the proteins, converting in one regular band into two bands on a gel (thereby making it hard to judge whether the correct proteins are expressed).
5. Film exposure time has to be adjusted (from 1 h to several days) so that the appropriate exposure is obtained. The criterion is that you can see the desired bands clear enough. Or you can use an imager to obtain the appropriate exposure through computer imaging.
1. Luo, W., Kuwada, T. S., Chandrasekaran, L., Zheng, J., and Tanzer, M. L. (1996) Divergent secretory behavior of the opposite ends of aggrecan. J. Biol. Chem. 271, 16,447-16,450.
2. Zheng, J., Luo, W., and Tanzer, M. L. (1998) Aggrecan synthesis and secretion: a paradigm for molecular and cellular coordination of multiglobular protein folding and intrac-ellular trafficking. J. Biol. Chem. 273, 12,999-13,006.
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