The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform
- PBS (phosphate buffered saline), without calcium and magnesium, 1×, pH 7.4 (Fisher Scientific, cat. #MT21040CV, Waltham, MA)
- Lysis buffer (see Recipes, made fresh immediately before homogenization and stored on ice)
- Pierce™ Protease Inhibitor Tablets, EDTA-Free (Thermo Scientific™, cat. #A32965, Waltham, MA)
- 1.5 ml Micro Centrifuge Tube, Sterile (Cell Treat, cat. #229443, Peperell, MA)
- Conical Tubes, 15 ml (Invitrogen™, cat. #AM12500, Carlsbad, CA)
- 70% ethanol
- Pierce™ BCA Protein Assay Kit (Thermo Scientific™, cat. #23225, Waltham, MA)
- Costar 96-Well Flat-Bottom EIA Plate (Bio-Rad, cat. #2240096, Hercules, CA)
- Collagen I, Rat Tail (Purified Protein, 3 mg/ml) (Gibco®, cat. #A10483-01, Carlsbad, CA)
- 4× Laemmli Sample Buffer (Bio-Rad, cat. #161-0747, Hercules, CA)
- Novex™ Tris-Glycine Native Sample Buffer (2×) (Invitrogen™, cat. #LC2673, Carlsbad, CA)
- β-mercaptoethanol (Fisher BioReagents®, cat. #BP176-100, Fair Lawn, NJ)
- Lysis buffer (see Recipes)
- Sample/protein of interest
- Western Blotting Filter Paper, 7 cm × 8.4 cm (Thermo Scientific™, cat. #84783, Waltham, MA)
- 30% Acrylamide/Bis Solution 29:1 (Bio-Rad, cat. #1610156, Hercules, CA)
- Tris-Base (Fisher Bioreagents®, cat. #BP152-5, Fair Lawn, NJ)
- 6 N HCl (FisherChemical, cat. #SA56-500, Fair Lawn, NJ)
- Sodium dodecyl sulfate (SDS), white powder (Fisher BioReagents™, cat. #BP166-500, Fair Lawn, NJ)
- Ammonium Persulfate (Sigma-Aldrich, cat. #A3678-25G, St. Louis, MO)
- TEMED (Bio-Rad, cat. #1610800, Hercules, CA)
- SureCast™ Gel Handcast Station (Invitrogen™, cat. #HC1000, Carlsbad, CA)
- SureCast™ Glass Plates (Invitrogen™, cat. #HC1001, Carlsbad, CA)
- SureCast™ Gel Spacer (Invitrogen™, cat. #HC1003, Carlsbad, CA)
- 1.0 mm 10-Well, 12-Well or 15-Well Combs (Novex®, cat. #NC3010, NC3012, NC3015, Carlsbad, CA)
- 2-Propanol (also known as isopropyl alcohol) (Fisher Chemical, cat. #A416-500, Fair Lawn, NJ)
- Novex™ 4%–12% Tris-Glycine Mini Gels, WedgeWell™ format, 10-well (Invitrogen™, cat. #XP04120BOX, Carlsbad, CA, store at 4°C for up to 1 year)
- NuPAGE™ 4%–12% Bis-Tris Protein Gels, 1.5 mm, 10-well (Invitrogen™, cat. #NP0335BOX, Carlsbad, CA, store at 4°C for up to 1 year)
- iBright™ Pre-stained Protein Ladder (Invitrogen™, cat. #LC5615, Waltham, MA)
- NativeMark™ Unstained Protein Standard (Novex®, cat. #LC0725, Carlsbad, CA)
- Tris base (Fisher BioReagents™, cat. #BP152-5, Fair Lawn, NJ)
- Glycine (Fisher Bioreagents™, cat. #BP381-5, Fair Lawn, NJ)
- Sodium dodecyl sulfate (SDS) (Fisher BioReagents™, cat. #BP166-500, Fair Lawn, NJ)
- 1× Tris-glycine running buffer (with or without SDS) (See Recipes) NuPAGE™ MOPS SDS Running Buffer (20×), diluted to 1× with diH20 (Invitrogen™, cat. # NP0001, Carlsbad, CA)
- Gel Loading Tips 1–200 µl (Fisherbrand™, cat. #02-707-181, San Diego, CA)
- Pierce™ Classic IP Kit (ThermoFisher™, cat. #26146, Rockford, IL)
- Anti-Collagen I, polyclonal rabbit (Abcam, cat. #ab34710, Cambridge, MA)
- Tris, 1.0 M buffer soln., pH 9.5 (Alfa Aesar™, cat. #AAJ62084K2, Ward Hill, MA)
- 1× Tris-glycine transfer buffer (See Recipes, store at 4°C)
- AmershamTM ProtranTM 0.2 µm NC Nitrocellulose Blotting Membrane (GE Healthcare Life Science, cat. #10600011, Germany)
- Western Blot Roller (Thermo Scientific™, cat. #84747, Waltham, MA)
- Excelta™ Plastic tweezers (Fisher Scientific, cat. #17-467-347, Fair Lawn, NJ)
- SureCast™ Multi-use Tool (Invitrogen™, cat. #HC1010, Waltham, MA)
- Ponceau S solution (Sigma-Aldrich, cat. #P7170-1L, St. Louis, MO)
- Hanks’ Balanced Salt Solution (HBSS), 1× (Gibco® by Life Technologies, cat. #14175-079, Grand Island, NY)
- Anti-Collagen, type 1, monoclonal mouse (Sigma-Aldrich, cat. #C2456, St. Louis, MO)
- Beta Tubulin Polyclonal Antibody, (ThermoFisher™, cat. #PA1-16947, Rockford, IL, used at 1:1000)
- IRDye® 800CW Goat Anti-Mouse (LI-COR®, cat. #925-32210, Lincoln, NE)
- IRDye® 800CW Goat Anti-Rabbit (LI-COR®, cat. #926-32211, Lincoln, NE)
- IRDye® 680RD Goat Anti-Rabbit (LI-COR®, cat. #926-68071, Lincoln, NE)
- Bovine Serum Albumin (BSA), Fraction V, Cold-ethanol Precipitated (Fisher BioReagents™, cat. #BP1605-100, Fair Lawn, NJ, store at 4°C)
- Tween® 20 (Sigma Aldrich, cat. #P1379, St. Louis, MO, very viscous liquid so pipette very slowly to not draw any air)
- Black Western Blot Incubation Boxes (LI-COR®, cat. #929-97201, Lincoln, NE)
- Lysis buffer (made fresh daily): Pierce protease inhibitor, EDTA-Free 1 tablet, PBS 50 ml, disssolve by vortexing
- 10% APS (made fresh daily): Ammonium persulfate 0.10 g, diH2O 1 ml, dissolve by vortexing
- 1.5 M Tris-HCl, pH 8.8 (150 ml): Tris base 27.23 g, diH2O 80 ml, adjust to pH 8.8 with 6 N HCl, diH2O to 150 ml
- 0.5 M Tris-HCl, pH 7.6 (100 ml): Tris base 6.00 g, diH2O 60 ml, adjust to pH 7.6 with 6 N HCl, diH2O to 100 ml
- 10% SDS (10 ml): SDS 1.00 g; diH2O 9 ml, dissolve with gentle stirring, diH20 to 10 ml
- 10× SDS-PAGE running buffer (1 L): Tris base 30.30 g, glycine 144.10, SDS 10.00 g, diH2O to 1 L. For 1×, mix 100 ml with 900 ml diH20.
- 10× native PAGE running buffer (1 L): Tris base 30.30 g, glycine 144.10 g, diH2O to 1 L. For 1×, mix 100 ml with 900 ml diH20.
- 10× PAGE transfer buffer (1 L): Tris base 105.0 g, Glycine 27.5 g, diH2O to 1 L. For 1×, mix 100 ml with 800 ml cold diH20 and 100 ml methanol.
- 10× TBS (1 L): Tris Base 24.2 g, NaCl 80 g, diH2O to 1 L. For 1×, mix 100 ml with 900 ml diH20.
- 1× washing buffer (TBST): 10× TBS 100 ml, Tween 20 1 ml, diH2O to 1 L
- Blocking buffer (5% BSA in TBST): 1× washing buffer (TBST) 100 ml, BSA (in 4°C fridge) 5 g. Allow to sit at 4°C for ~15 min to mix before using.
- Ponceau S staining buffer, 1:10 (50 ml): Ponceau S solution 5 ml, dH2O 45 ml
- –20°C freezer
- –80°C freezer
- Hot Plate (set to 100°C)
- Homogenizer VDI 12 (VWR™, cat. #431-0125, Radnor, PA)
- 2 VWR™ Platform Rocking Shaker 100 115V (Store one at RT and one at 4°C, VWR™, cat. #VWRU40000-300)
- Eppendorf® Microcentrifuge 5415 D placed in a 4°C refrigerator (Sigma-Aldrich, cat. #Z604062, St. Louis, MO)
- Shaking 37°C incubator
- PowerEase™ 300W Power Supply (Thermo Fisher Scientific™, cat. #PS0300, Waltham, MA)
- iMark™ Microplate Absorbance Reader (Bio-Rad, cat. #1681130, Hercules, CA)
- SureCast Handcast System (Invitrogen™, cat. #HC1000S, Waltham, MA)
- Mini Gel Tank (Invitrogen™, cat. #A25977, Waltham, MA)
- Mini Blot Module (Invitrogen™, cat. #B1000, Waltham, MA)
- Odyssey Classic Infrared Imaging System (LI-COR®, cat. #980-11174, Lincoln, NE)
1.Keep tissue in a sterile 15 ml conical tube on ice.
2.Prepare fresh lysis buffer and keep on ice.
3.Add 1.0 ml lysis buffer to the conical tube with the tissue and allow to sit for 15 min.
4.Assemble the homogenizer and sterilize by rinsing 3 times in 70% ethanol, then 2 times in PBS.
5.Turn on the homogenizer for a maximum of 5–10 s at a time to homogenize the tissue without overheating. Keep samples on ice during this step.
6.Once the sample is completely homogenized and in a mostly liquid-like state, transfer the sample to a sterile Eppendorf tube.
7.Rotate samples in a rotating rack for 15–30 min at 4°C.
8.Centrifuge at 12000 RPM for 15 min at 4°C.
9.Pipette off the supernatant into a sterile Eppendorf tube.
10.Follow Pierce™ BCA Protein Assay kit’s instructions.
11.Use the acquired protein concentration to calculate the number of µl of protein sample containing 20 µg total protein to be loaded into each well.
12.Preparation of separating gel:
12.1.Assemble glass plates with rubber divider in casting stand (ensure clean by washing first with soap & water, then 70% ethanol) and tilt the casting stand backward.
12.2.In a 50 ml conical tube, combine the following reagents for an 8% Tris-glycine gel (include SDS for SDS-PAGE; exclude SDS for NATIVE-PAGE and replace with diH20). Add TEMED last (dH2O 9.3 ml, 30% acrylamide 5.3 ml, Tris-HCl (1.5 M) 5.0 ml, SDS (10%) 0.2 ml, 10% APS (fresh) 0.2 ml, TEMED 0.012 ml).
12.3.Gently mix the tube by inverting slowly to avoid introducing air bubbles as oxygen inhibits polymerization.
12.4.Slowly transfer approximately ~8 ml of the solution or until it reaches the “fill line” on the plate and no more than 10 ml, into the gap between the glass plates to avoid forming gas bubbles using a serological pipette.
12.5.Top the gel off with a layer of isopropyl alcohol to limit contact with air and to remove air bubbles that may have formed when pouring the gel.
12.6.Allow the gel to polymerize (20–30 min).
13.Preparation of stacking gel:
13.1.Use filter paper to soak up the isopropyl alcohol that is above the separating gel.
18.104.22.168 In a 50 ml conical tube, combine the following reagents for an 8% Tris-Glycine Gel (include SDS for SDS-PAGE, or exclude SDS for NATIVE-PAGE and replace with diH20). Add TEMED last (dH2O 5.5 ml, 30% Acrylamide 1.3 ml, Tris-HCl (0.5 M) 1.0 ml, SDS (10%) 0.08 ml, and 10% APS (fresh) 0.08 ml TEMED 0.008 ml). 13.3 Gently mix the tube by inverting slowing (only a few times).
13.3.Slowly transfer approximately 3 ml of the solution into the gap between the glass plates to avoid forming gas bubbles.
13.4.Insert the comb at a slight angle and allow the gel to polymerize for about 20 min. If bubbles form around the comb, remove it and replace it again.
14.Turn on the heat plate.
15.Thaw protein samples on ice.
16.If the protein concentration of one’s sample is known, proceed to next step. If not, follow “Protein quantification.” For nonreducing conditions, steps 17–23 can be altered to exclude the reducing agent (BME).
17.Calculate the combined amounts of [sample buffer + BME] and [protein sample + lysis buffer] based on the desired total volume loaded into each tube (this protocol recommends 20 µl) and concentration of the sample buffer. For 4× sample buffer: combine 1 part [4× sample buffer + BME] with 3 parts [protein sample + lysis buffer]. Example: Total µl = 20 µl; 1/4 (20) = 5 µl = [sample buffer + BME]; 3/4 (20) = 15 µl = [sample + dilutant].
18.Calculate the individual amounts of sample buffer and BME necessary to make the combination calculated in step 17. This protocol recommends a 10% BME in sample buffer solution, which can be made by combing 1 part BME with 9 parts sample buffer. Example: 1/10 (5) = 0.5 µl = BME; 9/10 (5) = 4.5 µl sample buffer.
19.Calculate the individual volumes of protein sample and lysis buffer necessary to make the combination calculated in step 17. Example: 15 µl−Vsample = 15−3.62 = 11.38 µl lysis buffer.
20.Create a table as illustrated below (Table 1), including the numbers assigned to each lane.
21.Place the cassette clamps into the electrophoresis chamber, insert the gel(s), clamp, fill with running buffer to fill line, and remove the gel comb slowly and straight up.
22.Prepare individual solutions for each well in separate Eppendorf tubes. Add [BME + sample buffer] last.
23.Vortex and spin down the samples.
24.Boil the samples (if desired) at 100°C for 5 min.
25.Load 2 µl of iBright Fluorescent Marker into 1 or more lane.
26.Load the protein sample solutions into the other lanes.
27.Run the gel on constant mode at 125 V for handcast gels, 225 V for Novex 4%–12% Tris-Glycine precast gels, OR 200 V for NuPAGE™ 4%–12% Bis-Tris Protein Gels. Once the settings are correct and lid is secure, one can then hit “run”.
28.When the gel is finished running, turn off the power supply and remove the lid.
29.Remove cassette, separate the 2 sides of the gel (be careful not to rip the gel), nick the top left corner of the gel, and trim the top and bottom using the SureCast Multiuse Tool.
|Lane||Conditions||VSample (µl)||VLysis buffer (µl)||VBME (µl)||V4× sample buffer (µl)||VTotal (µl)|
|2||BME + boiled||3.62||11.38||0.5||4.5||20|
|3||BME + Not boiled||3.62||11.38||0.5||4.5||20|
|4||No BME + boiled||3.62||11.38||0||5||20|
|5||No BME + Not boiled||3.62||11.38||0||5||20|
|6||+-Control: BME + boiled||2||13||0.5||4.5||20|
|7||+-Control: BME + not boiled||2||13||0.5||4.5||20|
|8||+-Control: No BME + boiled||2||13||0||5||20|
|9||+-Control: No BME + not boiled||2||13||0||5||20|
30.Prepare 250 ml of 1× PAGE transfer buffer containing 10% methanol (see Recipes).
31.Place the gel in 1× transfer buffer to allow equilibration and washing of the running buffer salts to come off.
32.Soak 2 pieces of filter paper in 1× transfer buffer in a clean container.
33.Soak two sponge pads very thoroughly in 1× transfer buffer in a clean, separate container from everything else.
34.Cut a piece of nitrocellulose membrane to the size of the filter paper and in a third container, soak it in 1× transfer buffer. Be sure to nick the upper left hand corner.
35.Place the cathode core (-) side down first on a flat surface. Fill with ~10 ml of 1× transfer buffer.
36.Squeeze one sponge pad to remove excess transfer buffer and place in the cathode core.
37.Place one presoaked piece of filter paper on top of the sponge pad. Use blotting roller to remove any air bubbles.
38.Place pretrimmed and rinsed gel carefully on top of the filter paper using the gel knife.
39.Briefly wet the membrane in clean 1× transfer buffer and place over the gel using clean tweezers.
40.Place the second piece of presoaked filter paper on top of the membrane. Use blotting roller to remove any air bubbles. Do this carefully to not disrupt the sandwich already built.
41.Squeeze the second sponge pad to remove excess transfer buffer and place in the filter paper.
42.Place the anode core (+) on top of the sandwich, closing the module.
43.Place the entire module with cathode core (-) facing front into one of the chambers in the mini gel tank. Make sure the cassette clamp has been removed after running of the gel.
44.Fill the module core with 1× transfer buffer until the fill line. Do not over fill.
45.Add fresh 1× transfer buffer, about 225 ml, or dH20, to the outside chamber to the cathode on the tank.
46.With the power supply OFF, place lid firmly onto the tank base and plug the electrode cords into the corresponding holes on the power supply (red to red, black to black).
47.Turn ON the power supply and set to a constant voltage of 5 V. Transfer for 150 min.
48.Once the transfer is complete, turn the power supply to OFF.
49.Carefully dissemble the module core using care to not tear the membrane.
50.Using clean tweezers, place the membrane in a clean container with dH20 to rinse for at least 5 min to remove any solutions left on from the transfer.
51.To check transfer: cover the entire upper surface of the membrane with Ponceau S staining buffer and incubate until bands are visible (approximately 1 min). The Ponceau buffer may be collected and reused up to 10 times.
52.If not done earlier, nick top left corner of membrane with a clean razor and trim excess membrane.
53.Rinse the membrane with 1× HBSS until the background is clean.
54.Wash the membrane twice with distilled water for 5 min while preparing for blocking & incubating.
Membrane blocking & antibody incubation
55.Thaw antibodies on ice.
56.Incubate the membrane with 5 ml blocking buffer for 1 h at 4°C with gentle shaking in a black western incubation box. Discard solution.
57.Dilute the primary antibody to the desired concentration in 5 ml blocking buffer. Sigma C2456 primary antibody was used at a dilution ratio of 1:2000, equivalent to 2.5 µl C2456 in 5 ml blocking buffer.
58.Incubate the membrane with the diluted primary antibody overnight at 4°C with gentle shaking. Discard solution.
59.Wash the membrane 4× for 5 min at room temperature with 15 ml TBST and gentle shaking, discard solution after every wash.
60.Dilute the secondary antibody to the desired concentration in 5 ml blocking buffer. This protocol recommends using IR-Dye 800 Goat Anti-mouse at a dilution ratio of 1:20000, equivalent to 0.25 µl secondary antibody in 5 ml blocking buffer.
61.Incubate the membrane with the diluted secondary antibody for 1 h at room temperature with gentle shaking. Discard solution.
62.Wash the membrane 4× for 5 min at room temperature with 15 ml TBST and gentle shaking, discard solution after every wash.
63.Discard the last wash and add a little diH2O. Membrane is ready to be scanned on the Odyssey Classic Infrared Imaging System Imager (Protein Visualization).
64.Mammalian cell lysis
64.1.If you have your tissue lysate already, proceed to “Step 65” (as we did), or choose a protocol under “Step 64” that fits what type of lysate you are trying to achieve (Protocol I or II).
65.Pre-clear lysate using the control agarose resin
65.1.Instead of 1 mg of lysate, 500 µg of muscle lysate was used. Everything else remained the same.
66.Preparation of immune complex
66.1.For the monoclonal mouse antibody (Sigma) 2µl was loaded into each corresponding tube.
66.2.For the polyclonal rabbit antibody (Abcam) 1 µl was loaded into each corresponding tube.
66.3.300 µl of our Lysis buffer was used to dilute the antibody/lysate solution.
66.4.Incubation occurred overnight at 4°C.
67.Capture of the Immune complex
67.1.The alternative wash buffer (20× TBS buffer) was used at a 1× concentration (diluted with diH20) because detergent-free is important to our samples.
68.Elution of the Immune complex
68.1.The final product from “step 67” was split into 4 tubes to analyze the four different conditions via western blotting: Boil/BME, No Boil/BME, Boil/No BME, and No Boil/No BME.
68.2.Sample-buffer elution: Boil/BME conditions were implemented here. BME was used instead of DTT. This was performed after the Low-pH elution was done.
68.3.Low-pH elution: No Boil/BME, Boil/No BME, and No Boil/No BME conditions were done using this method and 4× Leammli buffer (Bio-Rad) was used. Three elutions were done and the optional step of neutralizing each elution with 1 M Tris, pH 9.5 was performed.
69.Use the Odyssey Classic Infrared Imaging System and Image Studio™ to capture an image of your finished membrane. Set conditions to: U 169 µm, Q high, F 0.0 mm, and intensity 5. Clean the surface with diH2O and wipe with a kimwipe.
70.Place membrane on screen using plastic tweezers.
71.Use mini roller to roll out any air bubbles.
|Collagen type 1 Disulfide bonded collagen 1/Intact triple helix (~250 + kDa)||
|Procollagen I subtypes (~180 to 200 kDa)||
|Mature collagen α1 (I) (~130 kDa)||
|Mature collagen α1 (I) (~130 kDa)||
|No signal||Concentration of the primary or secondary antibody is too weak||Use a higher concentration of primary antibody—success has been demonstrated with Sigma #C 2456 at a dilution ratio of 1:1000–1:2000|
|Insufficient antigen||This protocol recommends loading 20 μg of total protein per well, but up to 30 μg should work; if collagen bands still do not present, it is possible that the tissue itself does not contain substantial amounts of collagen|
|Poor transfer of collagen to nitrocellulose membrane||A longer transfer with a lower voltage can be used|
|High background||Insufficient blocking of membrane||Optimal blocking of non-specific binding was demonstrated using 5% BSA in TBST, but goat milk serum may also be used|
|Primary antibody concentration was too high||Decrease the dilution ratio of the antibody and incubate for longer|
|Incubation temperature is too high||Incubate the membrane in blocking buffer and primary antibody at 4°C|
|Secondary antibody is giving unexpected bands||Make sure a secondary only probing is done without primary antibody to verify that the bands are not due to secondary antibody nonspecific binding|
|Intensity is turned up too high on the Licor Imager||Reduce the intensity so that the background is reduced; if the intensity is too high background bands, spots, etc., can start to appear|
|White spots on the blot||Trapped air bubbles during transfer||Make sure to use a mini-blot roller to carefully remove bubbles when assembling the transfer|
|Overheating during transfer||Place gel tank in ice or in a coldroom for the duration of the transfer; and or reduce the voltage and increase transfer time|
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