Western Blot analysis

The procedure is developed for use at the Department of Clinical Research, Hvidovre Hospital, to detect specific protein(s) in a given sample.

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Procedure:

MATERIALS

4x NuPAGE® LDS Sample buffer (Invitrogen; NP0008)

Various parts of the XCell SureLock™ Mini-Cell

NuPAGE® Novex Bis-Tris Gels (consult Invitrogen web site for the reference numbers)

20x NuPAGE® MOPS SDS Running Buffer (Invitrogen; NP0001-02)

Markers for SDS-PAGE (MagicMark™ XP Western Protein Standard, Invitrogen, LC5602; Novex® Sharp Protein Standard, Invitrogen, LC5800)

Filter paper

Amersham Hybond™-P (GE Healthcare; RPN1416F)

Blotting pads

Primary and secondary antibodies

Signal West Femto Maximum Sensitivity Substrate (Pierce, 34095).

Furthermore, the contents of wash-, blocking-, transfer-, and running buffer are required (see below).

LIST OF BUFFERS

Running buffer

• 50 ml of 20x NuPAGE® MOPS SDS Running Buffer (Invitrogen, NP0001-02)
• 1 L dH₂O

Transfer buffer

• 50 mL NuPAGE® Transfer buffer 20X (Invitrogen, NP0006-1)
• 20 % Ethanol
• 1 L dH₂O

Always pour alcohol in water!!! Start to add the 50 ml of NuPAGE® Transfer buffer 20X, fill with dH₂O until 700 ml. Then, add 200 ml of 96% Ethanol and fill up to 1 L with dH₂O.

Blocking buffer (5% BSA or skimmed milk, 1x PBS, 0,1 % Tween-20)

• 25 g skimmed milk powder or BSA powder
• 500 ml PBS, pH 7,4
• 500 ul of Tween-20

Wash buffer (1x PBS, 0,1 % Tween-20)

• 1 L PBS pH 7,4
• 1ml Tween-20

 Wear gloves ALL the time.

SAMPLES PREPARATION (The samples have to be on ice to avoid proteins degradation)

1. Thaw the samples on ice
2. Measure the protein concentration of each samples using Bradford (Sigma-Aldrich, B6916-500 ml) or BCA assay (Thermo Scientific; #23252)
3. Aliquot the necessary amount of each sample in 1,5 ml Eppendorf tubes
4. Choose the same final loading volume for all of your samples which need to be loaded in the same gel
5. Depending of the final loading volume you have chosen, dilute 1:4 the loading buffer (4x NuPAGE® LDS Sample buffer; Invitrogen; NP0008) in each sample
6. If you need strongest denaturing conditions, dilute 1:10 the reducing agent (NuPAGE® Sample Reducing Agent 10X; Invitrogen; NP0004) in each sample
7. Reach the final loading volume by adding the necessary volume of buffer (same buffer as your samples to avoid pH changes, e.g. RIPA buffer)
8. Heat the samples for 10 minutes at 70°C

SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)

1. Unwrap the gel and remove the white strip at the bottom of the gel. Then, put the gel in the Buffer Core of the Xcell II^TM SureLock Mini-Cell
   Place the gel so that the comb is facing the center of the Buffer Core. Put a second gel or a BufferDam on the other side and force the arrangement tightly into the Lower Buffer Chamber, thus creating an airtight center.
2. Dilute 1:20 the running buffer (20x NuPAGE® MOPS SDS Running Buffer for Bis-Tris Gels only; Invitrogen; NP0001-02) in deionized water (dH₂O). Pour the 1x running buffer in the middle chambers. Make sure that the center chamber is not leaking and then, fill the outer chambers with running buffer
3. Remove the comb and check that the wells were not damaged and doesn´t contain any residues of gel
4. Load 5 ul of MagicMark™ XP Western Protein Standard, 10 ul of Novex® Sharp Protein Standard and the samples. Then, connect the gel electronically using a voltage of 150V and a current of 100mA for 1 h 20 min (the electrophoresis is finish when the front of migration is at the bottom of the gel)
   Novex Sharp Prestained Standard (10 uL) allows to estimate the efficiency of the transfert. Contrary to the MagicMark^TM XP Western Protein Standard (not visible on the membrane), Novex Prestained Standard  marker won´t be visible on your membrane revelation.

TRANSFER OF PROTEINS TO A MEMBRANE

1. Cut out 2 pieces of filter paper in 8,5 X 7,5 cm and 1 Hybond-P PVDF membrane in 8 X 7 cm per gel to transfer. Soak the filter papers and the 8 Blotting pads in transfer buffer until needed
2. Remove the filter paper (blue) covering the cut out membrane on both sides
3. Put the PVDF membrane in pure methanol for 10 sec and then, wash 2x in dH₂O. Lastly, equilibrate the membrane for 10 minutes in transfer buffer
4. Remove the gel from the electrophoresis chamber and break off the disposable cassette surrounding it using the gel knife. Carefully cut off the stacking gel and the last colored bands appearing in the end of the gel.
   To keep track of the orientation of the gel, it is advisable to draw a “map” of the gel showing the color bands
5. In the Blot module, put the contents in the following order: 4 blotting pads – 1 filter paper – gel – membrane – filter paper – 4 blotting pads. Lastly, put on the lid.
   If you need to Transfer two gels in one Blot module, put the contents in the following order: 3 blotting pads – 1 filter paper – gel – membrane – 1 filter paper – 2 blotting pads – 1 filter paper – gel – membrane – 1 filter paper – 3 blotting pads
6. Put the Blot module in the chamber and fill it with 1x transfer buffer. Verify that it is not leaking. Then pour dH₂O in the outer chamber to reduce heating
7. Next, connect the WB-cassette electronically, at 150V and 150 mA for 2 hours.
   You can adapt the time of transfer depending on the size of the protein of interest
8. After transfer, open the Blot module. The membrane side in contact with the gel is the side where the proteins were transferred (protein side). Check that the Novex® Sharp Protein Standard Marker was well transferred on the membrane. Only touch the membrane at its corners to avoid damages of the protein side
9. Draw a symbol of your choice in the upper left corner on the protein side with a pen to keep track of the orientation
10. Wash 3x 5 min the membrane with wash buffer

IMMUNOBLOTTING (Antibodies incubation)

1. Put the membrane in 30 mL blocking buffer under rotation for 1 h at room temperature
2. Dilute the primary antibody in 5 ml of blocking buffer at the appropriate dilution in a 50 ml centrifuge tube. Carefully slide the membrane into the 50 ml centrifuge tube with the protein side facing inward the tube.
   If the antibody datasheet does not have a recommended dilution, try a range of dilutions (1:100-1:1000) and optimize the dilution according to the results
3. Incubate the membrane overnight at 4˚C under rotation
4. Wash 3x 5 min the membrane with wash buffer
5. Depending of the species of your primary antibody, dilute the corresponding HRP-conjugated (Horse Radish Peroxidase) secondary antibodies in 40 ml of blocking buffer at the appropriate dilution. Incubate for 1 h at room temperature under rotation
6. Turn on the chemiluminiscense apparatus.
7. Wash 3x 5 min the membrane with wash buffer

CLEANING EQUIPMENT

1. Wash everything with water and then rinse with dH₂O
2. Let dry (NOT with the electrophoresis equipment contaminated with Ethidium Bromide in the gel room but close to the sink in the PCR room)
3. Store equipment in the Western blot cabinet in the PCR room

DEVELOPMENT

1. To develop 1 membrane, mix 750 uL from each reagent of the Signal West Femto Maximum Sensitivity Substrate (Pierce, 34095) immediately before use
2. Dry slightly the membrane by posing one of it corner on tissues. Place the membrane on a transparent plastic plate and add drop wise and homogeneously the mixed revelation solution on the protein side of the membrane. Incubate for 5 minutes
3. Cover the membrane with cellophane. Gently remove potential air bubbles and excess of revelation solution by rolling a 15 mL centrifuge pipette on the covered membrane
4. Put the membrane inside the chemiluminiscense apparatus, and do the following on the computer: Open “Genvej til labworks” Þ Aquire Þ Video/digital. For the preview, set an exposure time of 30 seconds.
   Next, open Aquire Þ Darkroom: Set the focus to 56 % and zoom to 26 mm). Press “Start Preview”. When a picture window emerges, press the “Stop Preview” button. To set the brightness of the picture, open Edit and choose Display range. At the extreme right of the Display range window, a moveable line will appear. Move this line to the left until you have a clear picture.
   From this preview, estimate the appropriate time of exposure to have the best picture. Then, set this time in the Video/digital window and press Capture. When a picture emerges, set the brightness as previously explained.
5. Save your picture
   To have your picture in a format compatible with Windows programs, press Print screen. Then, open Paint and paste your “Print screen”. Select the area corresponding to your gel, cut the selection (Ctrl X) and open a new paint document (Ctrl N). Paste your selection into the new Paint document and save it.
   You can also use Microsoft Photoeditor which is able to open the saved file from the Labworks program. In Photoeditor, it is possible to save the picture in a more commonly used format.

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All users of the western blot analysis are responsible to follow this instruction.

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