Western Blot

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Western Blot Protocol - de Lange lab

Western Blot Protocol

Required Solutions 10x PBS 80 g NaCl 2 g KCl 14.4 g Na2HPO4 2.4 g KH2PO4 dissolve into 800 ml ddH2O Adjust pH to 7.4 qs to 1000 ml autoclave make a 1x working solution Buffer C (4ºC) 20 mM Hepes-KOH pH 7.9 (from 0.5 M stock) 0.42 M KCl (from 3 M stock) 25% glycerol (from 80% stock) 0.1 mM EDTA (from 0.5 M stock pH 8.0) 5 mM MgCl2(from 1 M stock) 0.2% NP40 (from 10% stock) Use autoclaved stocks for all ingredients. Store at 4ºC Add right before use to 10 ml: 10 µl 1 M DTT (store aliquots at -20ºC, thaw once) 50 µl 100 mM PMSF (in iso-propanol -20ºC) 1 µl 10 mg/ml Leupeptin (-20ºC) 1 µl 10 mg/ml Aprotinin (-20ºC) 10 µl 1 mg/ml Pepstatin (-20ºC) Or, replace the last three protease inhibitors with one mini Roche proteaseinhibitor tablet (# 11 836 153001) and add DTT and PMSF as above. For looking at phosphorylated proteins, add the following phosphatase inhibitors to Buffer C: Na-beta-glycerophosphate to 50 mM (stock 1 M) NaF to 1 mM (stock 0.4 M) Na-ortho-vanadate to 1 mM (stock 0.1 M) Buffer D 20 mM Hepes-KOH pH 7.9 100 mM KCl 25% glycerol 0.1 mM EDTA (cold) 1

Western Blot Protocol - de Lange lab Add right beforeuse, for 250 ml: 250 µl 1 M DTT 1.25 ml 100 mM PMSF 10x Ponceau S Ponceau S 2% (w/v) TCA 30% (w/v) Dilute to 1x before use (can be re-used many times). 4x Laemmli Buffer 4.4 ml 0.5 M Tris (pH 6.8) 4.4 ml Glycerol 2.2 ml 20% SDS 0.5 ml 1% Bromophenol Blue 0.5 ml Beta-ME Aliquot and store at -20ºC. Dilute to 2x before use. 10x Running Buffer 30.3 g (0.25 M) Tris Base 144 g (1.92 M) Glycine 10 g(1%) SDS or appropriate for concentrated stock qs 1000 ml ddH2O Dilute 1:10 with ddH2O. pH will be 8.3 10x Towbin’s Electrotransfer Buffer 30.3 g Tris Base 144 g Glycine qs 1000 ml ddH2O 1x Towbin’s 100 ml 10x stock 200 ml (20%) Methanol qs 1000 ml ddH2O 1 ml (0.02%) 20% SDS– optional

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Western Blot Protocol - de Lange lab Laemmli Buffer Cell Extracts Cells can be directly lysed into Laemmlibuffer as follows: 1. 2. 3. 4. Harvest cells by trypsination and count cells. Keep everything cold after this step. Spin cells in media for 5 min at 1,000 g at 4ºC. Aspirate off media and resuspend pellet in 1 ml cold 1x PBS. Transfer 106 cells to an eppendorf tube and spin in microfuge for 4 min at 4,000 g at 4ºC. Aspriate off PBS. 5. Suspend cells in 100 µl hot Laemmli buffer. 6. Heat for 5 min(80- 100ºC). 7. Shear DNA through a 28 1/2 gauge insulin syringe 5 times, or sonicate. 8. Heat to 80-100ºC for 5 minutes before loading. 9. Load 10 µl (105 cells) per lane. Cells can also be scraped into Laemmli buffer with a rubber policeman but this procedure does not allow for a cell count. Buffer C Whole Cell Extracts 1. Trypsinize cells (minimally one 6 well dish well or one 10 cm plate; keepcells subconfluent). Keep everything cold after this step; chill tubes and solutions on ice. 2. Wash once with medium containing serum (to inactivate the trypsin), 2 times with cold 1x PBS. Cells can be counted in one of these steps. 3. Resuspend cell pellet in 5x the pellet volume of buffer C (4ºC). 4. Incubate on ice for 30 min with occasional mixing (flicking the tube). 5. Spin 15K RPM(microfuge at max setting), 10 min at 4ºC. 6. Set some supernatant aside for a Bradford assay and quick freeze the sample or aliquots on dry ice and store at -80ºC. 7. Optional: Dialyze to 50 vol buffer D for 2 hours at 4ºC before freezing but in this case, spin dialyzed sample for 5 min in microfuge at 4ºC and transfer the supernatant to a new tube before freezing. SDS-PAGE Gel and Western Blot Run thegel according to the instructions below. Refer to SDS-PAGE Gels on page 7 for details. 1. Measure protein concentration in duplicate using Bradford using a BSA standard curve. Run up to 70 µg/lane (expect 300-400 µg protein from a subconfluent 10 cm plate). 2. Mix extract 1:1 with Laemmli buffer and heat to 80-100ºC for 5 min (don’t boil too long, proteins get destroyed). 3. Run on an SDS-PAGE...
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