A method for extracting intracellular proteins using a high-speed refrigerated centrifuge

1. Lysis with Trixon-100 or NP-40 lysis buffer;
2. Freeze-thaw lysis method;
3. Grinding method;
“The classic procedure is described in Molecular Cloning, Lecture 2: First, lyse the cells using RIPA buffer, then scrape them off, incubate on ice for 30 minutes, subject to sonication, and centrifuge at 4°C for 10 minutes.”
(1) Extraction of total protein from monolayer adherent cells:
1. Discard the culture medium and invert the flask onto absorbent paper to allow the paper to soak up the medium (or place the flask upright for a while to let any residual medium flow to the bottom of the flask, then use a pipette to remove it).
2. Add 3 ml of 4℃ pre-cooled PBS (0.01 M, pH 7.2–7.3) to each cell bottle. Gently shake the bottle horizontally for 1 minute to wash the cells, then discard the wash solution. Repeat this procedure twice more, for a total of three washes to remove the culture medium. After completely discarding the PBS, place the culture flask on ice.
3. Add 10 μl of PMSF (100 mM) to 1 ml of lysis buffer, mix thoroughly, and place on ice. (PMSF must be mixed with the lysis buffer only after it has been shaken until no crystals remain.)
4. Add 400 μL of lysis buffer containing PMSF to each cell culture flask, and incubate on ice for 30 minutes. To ensure thorough cell lysis, frequently shake the flasks back and forth.
5. After lysis is complete, use a clean scraper to gently scrape the cells onto one side of the culture flask (perform this step quickly). Then, using a pipette, transfer the cell debris and lysis buffer into a 1.5 ml centrifuge tube. (Perform the entire procedure on ice whenever possible.)
6. Centrifuge at 4℃ at 12,000 rpm for 5 minutes. (Pre-cool the centrifuge in advance.)
7. After centrifugation, carefully aliquot the supernatant into 0.5-mL centrifuge tubes and store them at -20℃.
(2) Extraction of total protein from the tissue:
1. Place a small piece of tissue into the spherical part of a 1–2 ml homogenizer, and use clean scissors to mince the tissue as finely as possible.
2. Add 400 μl of single-detergent lysis buffer (containing PMSF) to the homogenizer and homogenize. Then place the sample on ice.
3. After a few more minutes, continue grinding and then place it on ice. Repeat the grinding process several times to ensure the tissue is thoroughly minced.
4. After lysis for 30 minutes, use a pipette to transfer the lysate into a 1.5 ml centrifuge tube. Then, centrifuge at 12,000 rpm for 5 minutes at 4°C in a high-speed refrigerated centrifuge. Carefully collect the supernatant, aliquot it into 0.5 ml centrifuge tubes, and store at -20°C.
(3) Extraction of total protein from adherent cells treated with drugs:
Due to the effects of the drug, some cells have detached. Therefore, in addition to following the procedure outlined in (1), you should also collect the cells from the culture medium. The following describes the extraction of total cellular protein from the culture medium:
1. Transfer the culture medium into a 15 ml centrifuge tube and centrifuge at 2500 rpm for 5 min.
2. Discard the supernatant, add 4 ml of PBS, and gently pipette up and down to wash. Then, centrifuge at 2500 rpm for 5 min. After discarding the supernatant, repeat the washing step once more using PBS.
3. After washing the supernatant dry with a pipette, add 100 μl of lysis buffer (containing PMSF) and lyse on ice for 30 min. During the lysis process, gently flick the tube frequently to ensure thorough cell lysis.
4. Mix the lysate with the lysate from the culture flask, then centrifuge at 4°C and 12,000 rpm for 5 minutes. Transfer the supernatant into 0.5 ml centrifuge tubes and store at -20°C.
(2) Determination of Protein Content
(1) Prepare a standard curve
1. Take 1 mg/mL BSA from -20℃, allow it to melt at room temperature, and set aside for later use.
2. Take 18 1.5 ml centrifuge tubes, group them into sets of three, and label them as follows: 0 μg, 2.5 μg, 5.0 μg, 10.0 μg, 20.0 μg, and 40.0 μg.
3. Add the various reagents to each tube according to the table below.
0 μg 2.5 μg 5.0 μg 10.0 μg 20.0 μg 40.0 μg
1 mg/mL BSA – 2.5 μL, 5.0 μL, 10.0 μL, 20.0 μL, 40.0 μL
0.15 mol/L NaCl 100 μl 97.5 μl 95.0 μl 90.0 μl 80.0 μl 60.0 μl
4. After mixing thoroughly, allow the sample to stand at room temperature for 2 minutes. Then perform colorimetric analysis using a bio-spectrophotometer (Bio-Photometer, Eppendorf).
(2) Determine the protein content of the sample.
1. Take an adequate number of 1.5 ml centrifuge tubes and add 1 ml of Coomassie Brilliant Blue solution stored at 4°C to each tube. After letting the solution sit at room temperature for 30 minutes, it can be used for protein assays.
Take 100 μl of Coomassie Brilliant Blue solution plus 0.15 mol/L NaCl solution, mix thoroughly, and let it stand for 2 minutes to serve as the blank sample. Pour the blank into a cuvette and, following the procedure used to prepare the standard curve, measure the blank sample using the "blank" setting.
3. Discard the blank sample, rinse the colorimetric cuvette twice with anhydrous ethanol (0.5 ml each time), and then rinse once with sterile water.
Take one tube of Coomassie Brilliant Blue, add 95 μl of 0.15 mol/L NaCl solution and 5 μl of the protein sample to be tested. After mixing thoroughly, let the mixture stand for 2 minutes, then transfer it into a dried cuvette and press the “sample” button to measure the sample.
Note: For each sample measured, rinse the colorimetric cuvette twice with anhydrous ethanol and once with sterile water. You can also prepare multiple samples simultaneously and measure them together, which will significantly save time when analyzing a large number of protein samples. The results obtained represent the protein content in a 5 μl sample.
(3) SDS-PAGE Electrophoresis
(1) Cleaning the glass plate:
With one hand firmly gripping the glass panel, use the other hand to lightly scrub it with a bit of laundry detergent. After cleaning both sides, rinse thoroughly with tap water, then rinse again with distilled water and let it dry upright in a basket.
(2) Gluing and Sample Loading
1. After aligning the glass panes, place them into the clamp and secure them tightly. Then, vertically mount them onto the rack, ready for sealing with glue. (During operation, ensure that the two glass panes are perfectly aligned to prevent glue leakage.)
2. Prepare the separating gel. After adding TEMED, immediately mix thoroughly and then proceed with pouring the gel. When pouring the gel, use a 10 ml pipette to draw 5 ml of gel and carefully dispense it along the glass surface. Stop pouring once the gel surface reaches the midpoint of the green band. Then, add a layer of water on top of the gel; sealing the gel with a water layer will accelerate its polymerization. (At the beginning of pouring, you can pour relatively quickly, but slow down as the gel surface approaches the desired height. During the process, make sure the gel flows smoothly down the glass surface—this way, no air bubbles will form in the gel. When adding the water seal, do so very slowly; otherwise, the gel may get disturbed and deformed.) " S9 O. L+ `- T: E
3. When a refractive line appears between the water and the glue, it indicates that the glue has begun to set. Wait another 3 minutes to allow the glue to fully solidify, then carefully pour off the upper layer of water and use absorbent paper to thoroughly blot up any remaining moisture.
4. Prepare a 5% concentrated gel. After adding TEMED, immediately mix thoroughly and then pour the gel. Fill the remaining space with concentrated gel, then insert the comb into the concentrated gel. During pouring, ensure that the gel flows smoothly down the glass plates to prevent air bubbles from forming. When inserting the comb, make sure it remains perfectly horizontal. Since the gel shrinks in volume as it solidifies, reducing the sample-loading volume in the wells, you’ll need to frequently top up the gel on both sides during the concentration gel’s curing process. Once the concentrated gel has fully solidified, gently pull out the comb vertically, holding it by both sides with your hands.
5. Rinse the concentrated gel with water and place it into the electrophoresis tank. (The small glass plate should face inward, and the large glass plate should face outward. If you’re running only one gel, place a plastic sheet on the opposite side of the tank, with the printed side facing outward.)
After measuring the protein concentration, calculate the volume of the solution containing 50 μg of protein; this volume will be the loading amount. Transfer the loaded sample into a 0.5 ml centrifuge tube, then add 5× SDS sample buffer to achieve a final concentration of 1×. (The total loading volume should generally not exceed 20 μl; the maximum allowable volume per well is 25 μl.) Before loading, heat the sample in boiling water for 5 minutes to denature the proteins.
7. After adding sufficient electrophoresis buffer, begin preparing to load the samples. (The electrophoresis buffer must at least cover the small glass slide on the inner side.) Use a micropipette to aspirate the sample by gently touching the wall of the tube—make sure not to draw in any air bubbles. Insert the pipette tip into the sample-loading well and slowly add the sample. (Adding the sample too quickly may cause it to splash out of the well; likewise, the presence of air bubbles could also lead to sample overflow.) Before loading the next sample, rinse the pipette tip three times in the electrophoresis buffer in the outer chamber to prevent cross-contamination.
(3) Electrophoresis: The electrophoresis time is generally 1–2 hours, with a voltage of 80 V being optimal; 100 V can also be used. Stop the electrophoresis as soon as bromophenol blue just begins to migrate out of the gel, and proceed with membrane transfer.
(4) Transfer the membrane
(1) To transfer one membrane, you’ll need to prepare six pieces of filter paper measuring 7.0–8.3 cm and one piece of nitrocellulose membrane measuring 7.3–8.6 cm. Be sure to wear gloves when cutting the filter paper and the membrane, as proteins from your hands could contaminate the membrane. Before use, immerse the cut nitrocellulose membrane in water for 2 hours. (Use tweezers to gently place one edge of the membrane into a petri dish containing ultrapure water, ensuring that the membrane floats on the surface of the water—with only the lower side in contact with the water. This allows the entire membrane to become uniformly wet due to capillary action. If the membrane sinks into the water, an air film will form between the membrane and the water, preventing the membrane from absorbing water effectively.)
(2) Place the transfer clamp, two sponge pads, a glass rod, filter paper, and the soaked membrane into an enamel dish containing transfer buffer.
(3) Open the clip and keep the black side horizontal. Place a sponge pad on top, then use a glass rod to roll back and forth several times to remove any air bubbles inside. (While rolling with one hand, use the other hand to press down firmly on the pad to prevent it from shifting around.) Place three layers of filter paper on the pad—alternatively, you can stack three sheets of filter paper together first and then place them on the pad. Hold the filter paper steady with one hand, and use the glass rod with the other hand to roll out any remaining air bubbles.
(4) Before removing the gel, you must first pry off the glass slide. Be gentle when prying—lightly and repeatedly pry on both sides. After prying for a while, the glass slide will start to loosen gradually until it’s completely removed. (Be extremely careful when prying—the glass slide is very fragile and can easily crack.) After removing the small glass slide, gently scrape off the concentrated gel (as the concentrated gel can interfere with the procedure), taking care not to puncture the separating gel. Carefully peel off the separating gel and place it onto filter paper. Use your hands to align it precisely with the filter paper, then gently roll it with a glass rod to remove any air bubbles. Place the membrane over the gel, ensuring that it fully covers the entire gel surface (once the membrane is in place, do not move it again) and eliminate any remaining air bubbles. Cover the gel with three layers of filter paper and continue to remove any trapped air bubbles. Finally, place another sponge pad on top and press down several times to close the clamp. Throughout the entire procedure, work in the transfer buffer and keep rolling to remove air bubbles continuously. The filter papers on either side of the membrane must not come into contact with each other; if they do, a short circuit could occur. (The transfer buffer contains methanol, so be sure to wear gloves during the procedure and keep the lab door open to ensure proper ventilation.)
Place the clamp into the transfer tank, ensuring that the black side of the clamp faces the black side of the tank and the white side of the clamp faces the red side of the tank. During electrotransfer, heat will be generated; therefore, place a piece of ice on one side of the tank to help cool it down. Typically, use 60 V for 2 hours or 40 V for 3 hours. (6) After the transfer is complete, stain the membrane with 1× Coomassie Brilliant Blue staining solution for 5 minutes (while shaking on a decolorization shaker). Then, rinse the membrane thoroughly with water to remove any unbound dye, and you’ll be able to visualize the proteins on the membrane. Allow the membrane to air dry and set it aside for later use. / h3 P" o9 d8 n% U8 X. R
(5) Immune Response
(1) After wetting the membrane with TBS from bottom to top, transfer it to a petri dish containing blocking solution and incubate on a shaker at room temperature for 1 hour.
(2) Dilute the primary antibody with TBST to an appropriate concentration (in a 1.5 ml centrifuge tube). Tear off a piece of plastic wrap of suitable size and lay it flat on the experimental bench; moisten the four corners with water to keep the plastic wrap smooth. Add the antibody solution onto the plastic wrap. Remove the membrane from the blocking solution, blot off any residual liquid with filter paper, then place the membrane protein side down onto the antibody solution. Gently lift the four corners of the membrane to expel any trapped air bubbles. Incubate at room temperature for 1–2 hours, then wash twice in TBST at room temperature on a shaker, each time for 10 minutes. Finally, wash once more with TBS for 10 minutes. (3) Prepare the secondary antibody dilution using the same method as above, and bring it into contact with the membrane. Incubate at room temperature for 1–2 hours, then wash twice in TBST at room temperature on a shaker, each time for 10 minutes. Finally, wash once more with TBS for 10 minutes before proceeding with the chemiluminescence reaction.
(6) Chemiluminescence, developing, and fixing
Mix equal volumes of reagents A and B on a piece of plastic wrap. After 1 minute, place the membrane protein side down and ensure thorough contact with the mixture. After another minute, transfer the membrane onto a fresh piece of plastic wrap, remove any residual liquid completely, seal it tightly, and place it into an X-ray film holder.
In the darkroom, pour 1× developer and fixer separately into plastic trays. Under the red safety light, remove the X-ray film and use a paper cutter to trim it to an appropriate size—each side should be 1 cm larger than the corresponding dimension of the membrane. Open the X-ray film holder, place the X-ray film onto the membrane, and once positioned, do not move it. Close the X-ray film holder and start timing. Adjust the exposure time appropriately according to the intensity of the signal; typically, this ranges from 1 minute to 5 minutes. You can also choose different exposure times and repeat the process several times to achieve the desired result. After exposure is complete, open the X-ray film holder, remove the X-ray film, and immediately immerse it in the developer for development. As soon as distinct bands become visible, promptly stop the development process. The typical development time is 1–2 minutes (at 20–25°C); if the temperature is too low (below 16°C), you’ll need to slightly extend the development time. Once development is finished, immediately immerse the X-ray film in the fixer. The standard fixing time is 5–10 minutes, until the film becomes completely transparent. After rinsing off any residual fixer with tap water, allow the film to air-dry at room temperature.
It should be noted that when moving the film during development and fixing, try to hold it by one corner and avoid scratching the film with your fingernails, as this could affect the results.
Gel image analysis involves scanning or photographing the gel and using a gel image processing system to analyze the molecular weight and net optical density values of the target bands.