1) Need six 150 mm dishes of cells. Remove from incubator, bring to bench, aspirate off medium, and wash each dish two times (15 ml each wash) with room temperature PBS. Place cells on ice between the washes.
2) For later analysis of column eluate by avidin-peroxidase, cell surface proteins will need to be biotinylated. For sequence analysis, biotinylation can be omitted. To biotinylate, add 15 ml/dish of 50 µg/ml biotin (Pierce #21335 NHS-LC Biotin; prepare from freshly made stock [10 mg/ml in DMSO]). Incubate on ice for 90 min, aspirate biotin, then wash with (2) 5 ml/dish of PBS-glycine (0.56 gm glycine in 150 ml PBS).
3) Remove cells by tapping the side of the dishes during the PBS-glycine washes if biotinylating, or in PBS wash if not biotinylating. Collect washes into two 50 ml conicals, spin for 5 min at 1500 rpm (4°C), aspirate supernatant, combine the cell pellets into one 50 ml conical, resuspend in 25 ml of PBS-glycine if biotinylated, or in 1 ml of lysis buffer on ice for 25 min if not biotinylated. Spin PBS-glycine cells, aspirate supernatant, resuspend in 25 ml of PBS-glycine, respin, and resuspend in 1 ml of lysis buffer on ice for 25 min.
Lysis Buffer ddH2O 1.8 ml 1M Tris, pH 7.4 100 µl NaCl 11.6 mg MnCl2 (0.5 M) 20 µl PMSF (100 mM) 40 µl leupeptin (5 mg/ml) 4 µl peptstatin (5 mg/ml) 1.6 µl aprotinin (10 mg/ml) 2 µl octylglucoside (Sigma#O-8001) 0.117 gm* *or Triton X-100 40 µl
4) Transfer to two microfuge tubes and spin for 15 min at 4°C. Use supernatant to apply to columns. Prepare columns (have blank precolumn, SNpeptide column [and for purification/sequencing, a Con A column]) in step 3) by first draining, then equilibrating in 6 volumes of column running buffer. Following equilibration, columns should be 'preconditioned' with 5 column buffers of eluant buffer (ie. column running buffer containing 1 µg/ml heparin), then wash with 6 column volumes of column running buffer. Allow supernatant to flow through precolumn into Con A or SNpeptide column. Incubate 6 hr to overnight at 4°C.
Column Running Buffer ddH2O 165.6 ml 1 M Tris, pH 7.4 9.2 ml NaCl 1.1 gm 0.5 M MnCl2 1.8 ml PMSF (100 mM) 3.6 ml octylglucoside (Sigma#O-8001) 2.63 gm* *or Triton X-100 180 µl
5) Wash with 20 column volumes of column running buffer. Elute SNpeptide column with 5 ml column running buffer containing 1 µg/ml heparin or in which MnCl2 is replace with 10-20 mM EDTA. Elute Con A column with 5 ml column running buffer containing 0.4 M a-methylmannoside. Next wash columns with 5 column volumes of column running buffer containing 1 M NaCl, 5 column volumes of column running buffer and then the same volume of 50 mM Tris containing 0.02% Azide for storage (4°C). Be sure to keep column running without stop.
6) Subsequent analysis of eluant fractions can be performed following acetone precipitation of individual fractions, or spin concentration of all eluant fractions combined. For acetone precipitation (in 1.5 ml screw top tubes, add 300 µl of each fraction to 300 µl of -20°C 100% acetone, mix and store overnight at -20°C. Spin for 15 min at 4°C, pull/discard supernatant and add 1 ml/tube of 50% acetone (precooled to -20°C), mix and spin for 15 min at 4°C. Remove/discard supernatant. Use yellow tip to remove all remaining supernatant and let pellet dry for 1 hr at RT. Make pellet up in 40 µl of SDS (5%) gel sample buffer, boil for 5 min at 90°C, place 1 min on ice, spin for 1 min (RT), transfer to a fresh tube and load 15 µl/well on a 8% or 5-15% SDS PAGE gel (See ÔPouring and Running Protein Gels/ElectrotransferÕ). Run at 25 mAmp/gel. For spin concentration, add part of eluant to a Centricon-100 (preblocked by incubation in 1% BSA in PBS for 1 hr at 4°C; at end of block, spin for 15 min at 2500 rpm in SA600 rotor with rubber inserts [4°C]; then do a PBS wash). Spin for 30 min to 1 hr, discard flow through and more eluant and spin. Repeat until all has been concentrated to minimum volume. Add 1/6 volume SDS (2-5%) gel sample buffer, boil for 5 min at 90°C, place 2 min on ice, spin for 1 min (RT), and load all in one or two wide wells on a 8% or 5-15% SDS PAGE gel. Run at 25 mAmp/gel.
7) For biotinylated prep, electrotransfer from gel to Hybond-ECL nitrocellulose (Amersham#RPN 2020 D), as follows. Have blotting buffer prepared (can be reused a number of times; record each time used by marking label). Cut nitrocellulose to same size (or slightly larger) as gel. Have two pieces of Whatmann 2 MM paper cut to same size as gel. Have plastic tray containing transfer buffer and immersed in it in the following order: plastic cage ('+' side down), sponge (push out air bubbles), one piece of Whatmann paper and nitrocellulose filter. Turn off power to gel. Place gel plates on bench. Gently remove glass side. Notch bottom left corner. Discard stacking gel. Carefully invert gel onto nitrocellulose and use a spatula or gel spacer to encourage gel to separate from plate onto filter. Add onto gel in order: other Whatmann piece, sponge (remove bubbles), and other half of cage. Insert caged gel into blotting buffer and transfer (- from gel to + on filter) overnight at 60 mAmp (RT). For spin concentrated prep, remove gel and fix/stain in 0.08% Commassie blue in 25% isopropanol/10% methanol for 30 min (up to 2 hr). Destain in 10% acetic acid/10% isopropanol. Photograph. Band(s) corresponding to receptor is carefully cut out with a clean scapel blade, placed in an ependorf tube and stored at -20°C until ready for sequencing.
8) Stop transfer, remove cage into transfer buffer. Use black marker to outline orientation of gel on filter, and remove filter. Immerse filter in 50 ml of 37°C PBS-T (PBS containing 0.1% Tween-20; need 500 ml total per blot experiment) containing 2.5% milk for 1 hr at 37°C. Wash 1 x (50 ml) with PBS-T for 15 min, then 2 x (50 ml each) with PBS-T for 5 min each. Incubate in 50 ml of streptavidin-HRP (Amersham#RPN 1231; dilute 1/1000 in PBS-T) for 1 hr at RT on rotator. Wash 1 x 15 min and 4 x 5 min with PBS-T. Detect using ECL (in dark room, immediately before use, mix equal volumes of the two reagents, immerse filter for 1 min; cover filter with plastic wrap and detect with X-ray film).
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