Western Blot Protocol Protein isolation

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1 Western Blot Protocol Protein isolation A. Preparation of cell lysates. - Preparation of materials: -Dial the microcentrifuge temperature control setting to 4 C -Prepare a bucket of ice -Prepare lysis buffer * Add 1 protease inhibitor tablet/ 10 ml of Lysis Buffer prior to use ( ) 1. Collect cells (T25 flask=10 6 cells=1mg protein), spin 1500g x 5, discard supernatant. Transfer to Eppendorf. (Store C) Stored: C -decant any residual media in tube by inversion on paper towel. 2. Lyse the pellet with 100ul Lysis Buffer on ice for 10 min. (use 50ul for small pellet). Gently agitate w/ pipet or vortex at low speed. 3. Spin at rpm in a pre-cooled microcentrifuge for 10 min at 4 C. Transfer supernatant to new Eppendorf by careful pipetting. Dilute 1:10 with PBS (approx. 900 ul PBS). If 50ul LB used then 450ul PBS 4. Determine the protein concentration---microtiter Plate Protocols Mix 1ml Comassie Blue (CB) with 4ml ddhoh. Prepare five pre-made dilutions of a protein standard (BSA): 0,125,250,500,750,1000 ug/ml. (PIERCE std dilutions (stored in 4 C)). *Gently flick standard tubes to suspend proteins. Add 200 ul of diluted Comassie Blue to appropriate wells of microtiter plate. Pipet 10ul of each standard and sample solution (1:10) 1 into separate wells on 96 well plate containing 200 ul CB Mix thoroughly. Avoid Bubbles. Duplicate. Incubate at room temperature 5 min. Measure absorbance at 595 nm. 2 Use typical standard curve to calculate the concentration of samples. 3 Use closest std to calc concentration of all samples. 5. Take 50ul sample and mix with 50ul of 1X Laemmli sample buffer 4 (Add 50μl βme to 950μl sample buffer). Dilution factor: (1:10 / 2) 6. Boil for 5 min. 5 Cool at RT for 5 min. Spin to bring down condensation prior to loading gel. B. Running the gel. 1. Put NuPAGE Novex Bis-Tris Gel into Novex Mini Cell (Invitrogen) 2. Prepare 1X SDS Running Buffer by diluting 20X NuPAGE MES SDS Running Buffer. Fill the Upper and Lower Buffer Chamber with 1X SDS Running Buffer. 3. Load the appropriate concentration of protein sample and Marker on the wells of gel. (aspirate out gel parts with needle; only 15ul in 15well gels; 20ul in 10well gels) 4. Run conditions: Voltage: 186 V. Run 35 min. Expected Current: ma/gel (start); ma/gel (end) 5. After running the gel, use a knife to take out the gel, cut the portion of wells. 1 (200μl sample: 1800 μl PBS) = 1:10 dilution (200 in 2000 total) 2 See page 4. 3 See page 5. 4 (50 μl sample: 50 μl Laemmli Buffer) = 1:2 dilution (50 in 100 total) 5 Cover cap of tube with parafilm prior to boiling to prevent cap from popping during boiling 1

2 C. Semi Dry Western transfer protocol-assemble Sandwich 1. Prepare 100ml 1X NuPAGE transfer buffer: 5 ml 20X NuPAGE transfer buffer. (for 1 gel) 2. Prepare transfer membranes to the same dimensions as the gel. 3. Soak 2 blotting pads and transfer membrane with transfer buffer.(use tweezers) 4. Place a piece of pre-soaked blotting pad on the bottom of SEMI-DRY TRANSFER CELL (BIO-RAD), remove any trapped air bubbles. 5. Put the membrane on the pad, then put the gel on the membrane. 6. Place another blotting pad on top of the membrane. 7. Wet gel/membrane assembly with 1X NuPAGE Transfer buffer and remove any air bubbles. 8. Place the lid on the unit and connect the electrical leads to the power supply. Transfer condition: Voltage: 12-15V Transfer time: 30 min 9. Prepare Washing buffer and Blocking buffer. 10. When finished, carefully open the lid, use tweezers to take out the membrane, use pencil to mark the direction. Immerse membrane in Blocking buffer for 0.5 hour. D. Antibodies reaction 1. Incubate with primary antibody diluted in Blocking buffer (1:2000) for1-2hrs at RT. *- Save primary antibody (Ab); (ug/ml). 2. Wash 3 X 10 min with Washing buffer on shaker. 3. Incubate with secondary antibody diluted in Blocking buffer (1:2000) for 1 hr at RT. *- Save secondary antibody (Ab); (ug/ml). 4. Wash 3X 10 min with Washing buffer on shaker. 5. Add SuperSignal West Femto Substruct (1ml Stable Peroxide buffer & 1ml Lumino/Enhance Solution) to the membrane for 5 min at RT. Cover membrane to make dark. 6. Place membrane on plastic wrap. E. Detection 1. Adhere the membrane inside clear wrap to the bottom of a cassette. Rub out bubbles. 2. Exposure 1-5minutes. Total exposure time: min. Place a film on the membrane in the dark room for appropriate time. 3. Compare the band on the film with the marker on the membrane. Results F. Stripping membrane for additional blotting. 1. Place membrane in Restore Western Blot Stripping Buffer for 5-15 minutes at RT on shaker. Need just enough to cover membrane. 2. Wash 3x10 min with Washing Buffer, then Blocking Buffer 30min. 3. Continue above protocol for primary and secondary antibodies. G. Antibodies reaction Part Two 1. Incubate with primary antibody diluted in Blocking buffer (1:2000) for1-2hrs at RT. *- Save primary antibody (Ab); (ug/ml). 2. Wash 3 X 10 min with Washing buffer on shaker. 3. Incubate with secondary antibody diluted in Blocking buffer (1:2000) for 1 hr at RT. *- Save secondary antibody (Ab); (ug/ml). 4. Wash 3X 10 min with Washing buffer on shaker. 5. Add SuperSignal West Femto Substruct. 1ml Stable Peroxide buffer & 1ml Lumino/Enhance Solution, add to the membrane for 5 min at RT. Cover membrane to make dark. 6. Repeat Section E. 2

3 Results Buffer for Western: Lysis Buffer 50ml working stock: Final vol. to 50ml with ddh 2 O (approx. 47 ml) Reagent Amount 0.15M NaCl 1.5ml of 5M stock 5mM EDTA ph 8 0.5ml of 5mM stock 10mM Tris-HCl ph ml of 1M stock ph 7.4 Tween ml of 100x stock Protease Inhibitor Cocktail Tablet 1 tablet added just before use* * one tablet/10ml of Lysis Buffer 1. Comassie Blue Solution (1ml CB + 4ml ddhoh) 2. Laemmli sample buffer (heat stock soln, then 950ul+50ul βme = 2x sample buffer) 3. BSA 4. 20X NuPAGE MES SDS Running Buffer X NuPAGE transfer buffer 6. Primary antibody and Secondary antibody diluted in blocking buffer. 7. SuperSignal West Femto Substruct. Use Pico if signal too robust. Washing buffer Reagent Amount 0.15M NaCl x(5000mm)=500ml(150mm), x=15 10mM Tris-HCl ph7.5 x(1000mm)=500ml(10mm), x=5 0.05%Tween ml for 500ml Blocking buffer Add 5.0 gm of Milk Powder to 100ml of ddh 2 O in Erlenmeyer makes 5%. Use stir plate to mix. 3

4 Measuring the Absorbance: (Machine located in MSRB 167). 1. Turn on the TECAN machine (green light should appear in center front) 2. Log-On to the computer using Logon and password: ; 3. Open Magellan program a. Wizard List window will open. b. Tray holder will automatically open on front side of TECAN. c. Insert sample plate into reading slot. The orientation of the plate does not matter. d. Click plate in tab, then hit cancel tab. e. The plate readout will then display. 4. Click on Instrument a. From the drop down menu select Move plate and filter b. Under excitation/ Absorption select out tab. c. Filter tray will open, add appropriate filter to the slot. Filters kept in drawer underneath TECAN machine. 5. Click on Insert a. From the drop down menu select measurement parameters i. Under general tab select absorbance ii. Under plate tab browse and ensure NUNC96ft.pdf is chosen iii. Under measurement parameters set to desired wavelength 6. Click on Shaking a. Select time duration for shaking b. Select 5 seconds c. Click OK 7. Click on Instrument a. Click Start Measurement b. Give the file a name (yyyy/mm/dd-lastname) ( ) c. Click START 8. Machine will read the plate and display the results a. Print a copy of the readout to printer (if avail) otherwise, copy the readings. b. Leave the filter in the reader c. Log-Off the computer d. Turn off the TECAN 4

5 Plotting the Data: 1. Open Microsoft Excel sheet titled Formula for prot conc 2. In Column A enter the concentrations from the microtiter plate readout. 3. In Column B enter the corresponding Standard for each column in A. 4. Highlight the entire data section and graph an XY scatter plot. 5. Add a trendline and equation for the line by right clicking a single data point and selecting add trendline and selecting show equation. 6. From the equation of the line you can then use the OD readings from the samples as the x value. The y value will tell you the concentration of protein in the sample. The actual [protein] will be given by dividing by your dilution factor from step 4 of section A. 5

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