westernblotting12-general

westernblotting12-general - V.Woriax 9/05 BIO 3710-Cell...

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V.Woriax 9/05 BIO 3710 -- Cell Biology ---- 12% SDS-PAGE Preparation, Electrophoretic Transfer of Protein, and Immunodetection Purpose of Western Analysis : Separation of proteins, transfer to nitrocellulose and immunodetection of a specific protein (BSA, bovine serum albumin). Precautions: Please refer back to handout on the earlier set-up for performing an SDS-PAGE. REMINDERS : Acrylamide and bis-acrylamide are neurotoxins and must be handled carefully. Wear gloves, labcoat, and protective eyewear. Keep working chemicals on benchkote at all times. TEMED is highly flammable and volatile. Keep capped and take care in dispensing (using hood as necessary). Reference: Laemmli, UK (1970) Nature , 227 : 680-685 DAY I: A. Cast Gels 1. Clean glass plates with soapy water, blotting with paper towels. Use EtOH to dry completely. 2. Assemble glass plate sandwich according to manufacturer's instructions. Check for leakage. 3. Prepare 10% APS (ammonium persulfate) = 100 mg per 1ml of dH 2 O in a microfuge tube. 4. Assemble gel mix (add APS and TEMED just prior to use) 12 % Separator Gel 4 % Stacker 30 % Acrylamide/Bis 2.00 ml 333 μl 1.5 M Tris-HCl, pH 8.8 1. 25 ml ------ 0.5M Tris-HCl, pH 6.5 -------- 630 μl 10 % SDS 50 μl 25 μl dH 2 O 1.67 ml 1.4 ml 10 % APS 25 μl 25 μl TEMED 2.5 μl 2.5 μl 5. Add separator gel to opening between glass plates up to 1.8-1.9 cm from the top of the short plate, then add a thin layer (0.25-0.5 centimeter) of isobutanol and allow gel to harden 15-20 minutes. Once the gel has solidified, pour off the isobutanol. 6. Prepare stacker, placing it on top of separator gel, then insert comb into stacker gel. Position comb and allow stacker to harden; place polymerized gel in a covered container. Store gels moist at room temperature or refrigerated until next laboratory period. B. Prepare Running Buffer: Make 1X Running Buffer (250 ml)- Dilute 10 X stock running buffer to 1X running buffer Day II: Macromolecules such as proteins or nucleic acids can be separated in a matrix such as acrylamide or agarose. However, the molecules are then trapped in the matrix of the gel and cannot be detected by antibodies or oligonucleotides (a specific sequence of nucleotides) used as probes. In these cases, the probes and molecules are too large to diffuse through the gel to bind
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westernblotting12-general - V.Woriax 9/05 BIO 3710-Cell...

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