Materials and reagents

Biological materials

1. C2C12 cell line (KAC, Kyoto, catalog number: EC91031101-F0)

   The C2C12 cell line is an immortalized mouse myoblast cell line (ATCC, catalog number: CRL1772)

Reagents

1. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D6046-500M)

2. Penicillin/streptomycin solution (FUJIFILM Wako Pure Chemical Corporation, catalog number: 168-23191)

3. Fetal bovine serum (FUJIFILM Wako Pure Chemical, catalog number: 554-04855)

4. Glyceraldehyde (Nacalai Tesque, catalog number: 17014-81)

5. Phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺ [PBS(-)], 20× (LSI Medience, catalog number: PM102-PN)

6. 2-amino-2-hydromethyl-1,3-propanediol (Tris base) (Tris) (FUJIFILM Wako Pure Chemical, catalog number: 011-20095)

7. Urea (FUJIFILM Wako Pure Chemical, catalog number: 217-01215)

8. Thiourea (FUJIFILM Wako Pure Chemical, catalog number: 206-17355)

9. CHAPS (DOJINDO, Kumamoto, Japan, catalog number: 349-04722)

10. Methanol (FUJIFILM Wako Pure Chemical, catalog number: 131-01826)

11. Protease Inhibitor cocktail cOmplete Tablets EDTA-free, EASY pack (Roche, catalog number: 04-693-132-001)

12. BSA fraction IV (FUJIFILM Wako Pure Chemical, catalog number: 019-2329)

13. Bradford dye reagent (Takara Bio, catalog number: T9310A-1)

14. Skim milk for immunoassay (Nacalai Tesque, catalog number: 31149-75)

15. Tween-20 (GE Healthcare, catalog number: 17-1316-01)

16. Polyclonal anti-GA-AGE antibodies (purchased from Prof. Masayoshi Takeuchi, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, Uchinada, Japan; 920-0293).

    Note: Prof. Takeuchi successfully prepared the polyclonal antibody for TAGE, despite having limited information on the structure of TAGE [21]. Takeuchi et al. reported the hypothetical structure of TAGE in 2023 [24]. The antibody is preserved at -80 °C.

17. GA-AGE-BSA, 10 mg/mL (or TAGE-BSA, purchased from Prof. Takeuchi)

    Note: Despite limited information on the intra- and intermolecular structure of TAGE-BSA, the polyclonal anti-GA-AGE antibody (Reagent 17) developed by Prof. Takeuchi could be probed against the antigen recognition site in TAGE-BSA [21]. The GA-AGEs-BSA was dissolved in PBS and preserved at -30 °C.

18. Horseradish peroxidase (HRP)-conjugated molecular weight marker (Bionexus, catalog number: BNPM41)

19. Polyclonal goat anti-rabbit immunological HRP-conjugated antibody (DAKO, catalog number: REF0448)

20. ImmunoStar LD kit (FUJIFILM Wako Pure Chemical, catalog number: 292-69903)

21. Milli-Q ultrapure water

    Note: Purchased with an RFU554CA ultrapure water system (Advantech Toyo, Tokyo, Japan) at the Kanazawa Medical University.

Solutions

1. Medium for cell culture (see Recipes)

2. PBS(-) 1× (see Recipes)

3. Solution A (see Recipes)

4. Solution B (see Recipes)

5. Solution C (see Recipes)

6. Solution D (see Recipes)

7. 20 mg/mL BSA in Solution D (20 mg/mL BSA solution) (see Recipes).

8. Diluted BSA solution (see Recipes).

9. Diluted GA-AGEs-BSA solution (see Recipes)

10. HRP-conjugated molecular weight marker solution (see Recipes)

11. PBS-T (see Recipes)

12. 5% SM-PBS-T (see Recipes)

13. 0.5% SM-PBS-T (see Recipes)

14. Primary antibody solution (see Recipes)

15. Neutralized primary antibody solution (see Recipes)

16. Secondary antibody solution (see Recipes)

1. Medium for cell incubation

   Mix the reagents on a laminar flow hood.

   Note: Because this step can easily be performed in-house prior to the experiment, we do not provide a detailed description here. Complete information may be found in our previous studies [10–16]. Media can be stored in the general refrigerator (4 °C).

   Reagent	Volume	Recommended storage condition
   DMEM	450 mL	4 °C
   Penicillin/streptomycin solution	5 mL	-30 °C
   Fetal bovine serum	50 mL	-30 °C
   Total	505 mL




2. PBS(-) (1×)

   Room temperature refers to 22–28 °C.

   Reagent	Volume	Recommended storage condition
   PBS(-) (20×)	50 mL	Room temperature
   Milli-Q water	950 mL	Room temperature
   Total	1,000 mL




3. Solution A

   Using a 50 mL polypropylene centrifuge tube and serological pipet, dissolve Tris in Milli-Q water (10 mL) to prepare 1 mol/L (M) Tris solution. Solution A may be stored at -30 °C for 6 months.

   Note: The pH of the Tris solution was approximately 9.4. Only Tris powder was dissolved in Milli-Q-water, without adding other reagents (e.g., hydrochloride).

   Reagent	Quantity/volume	Recommended storage condition	Final concentration
   Tris	1.21 g	Room temperature	1 mol/L (M)
   Milli-Q water	10 mL	Room temperature
   Total	10 mL




4. Solution B

   Dissolve one tablet of protease inhibitor cocktail (cOmplete Tablets EDTA free) in Milli-Q water (2 mL) within a microcentrifuge tube; transfer the Milli-Q water to the tube using a 100–1,000 μL Gilson PIPETMAN. We recommend Solution B (2 mL) to be prepared and used immediately with every experiment. However, Solution B can be preserved at -30 °C for three months if it is reused. Approximately 100 μL of Solution B were preserved.

   Reagent	Quantity/Volume	Recommended storage condition	Final concentration
   Protease inhibitor cocktail	1 tablet	4 °C	1 tablet/2 mL
   Milli-Q water	2 mL	Room temperature
   Total	2 mL




5. Solution C

   Solution C contains 30 mM Tris, 7 M urea, 2 M thiourea, and 4% CHAPS. Mix these reagents in a 50 mL polypropylene centrifuge tube and add Milli-Q water to a final volume of 20 mL. Treat 1 M Tris with Milli-Q water using a 200–1,000 μL Gilson PIPETMAN.

   Note: The reagents are added to the centrifuge tube in no specific order. At room temperature, urea and thiourea are not easily dissolved in Milli-Q water; accordingly, we recommend using a vortex system (e.g., Vortex-Genie 2) at room temperature. We do not recommend dissolution in a 37 °C CO₂ incubator or water bath because urea and thiourea may produce cyanate or isocyanic acid at high temperatures [9]. Solution C can be preserved at -30 °C for three months, though we recommend preparing it immediately before use.

   Reagent	Quantity/Volume	Recommended storage condition	Final concentration
   1 M Tris	0.6 mL	Room temperature	30 mM
   Urea	8.40 g	Room temperature	7 M
   Thiourea	3.04 g	Room temperature	2 M
   CHAPS	0.80 g	Room temperature	4 w/v (%)
   Milli-Q water	20 mL	Room temperature
   Total	20 mL




6. Solution D

   Prepare Solution D by mixing Solutions B and C at a ratio of 1:9. Transfer Solution C using the 200–1,000 μL Gilson PIPETMAN. Transfer Solution D using a 10 mL serological pipette. Solution D contains 27 mM Tris, 6.3 M urea, 1.8 M thiourea, and 3.6 v/w (%) CHAPS.

   Note: We recommend dissolution using a vortex system (e.g., Vortex-Genie 2) at room temperature. Solution D can be preserved at -30 °C for three months, though we recommended preparing it immediately before use. Solution D was used as lysis buffer to prepare the cell lysate [8–17], which was prepared from C2C12 cells treated with glyceraldehyde [14]. Although “Modified Solution C” containing 30 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS, and 4% Solution B may also be used as lysis buffer [18–20,22], we recommend that Solution D be used because it contains sufficient urea, thiourea, and CHAPS to generate the carbamylated AGE proteins [8,9]. The cell lysates and unused Solution D should be preserved at -30 °C until later use in the slot blot experiment. The condition of the unused Solution D and the cell lysates should be arranged.

   Reagent	Volume	Final concentration
   Solution B	2 mL	10%
   Solution C	18 mL	90%
   Total (optional)	20 mL




7. BSA–Solution D mixture (20 mg/mL BSA solution)

   Dissolve 20 mg of BSA in Solution D (10 mL) within a 50 mL polypropylene centrifuge tube. Transfer Solution D using a 200–1,000 μL Gilson PIPETMAN.

   Note: BSA cannot be easily dissolved in Solution D; thus, we recommend using a vortex system (e.g., Vortex-Genie 2) at room temperature. The presence of undissolved BSA can be confirmed by the presence of a gelatinous substance. Dissolution by vortex should not be performed at high temperatures (e.g., 37 °C) [9].

   Note: We do not recommend commercial BSA for use in this experiment. An in-house solution should preferably be prepared. We believe that BSA fraction IV is suitable for the Bradford method in our protocol.

   Reagent	Quantity/Volume	Final concentration	Recommended storage condition
   BSA	20 mg	2.0 mg/mL (μg/μL)	4 °C
   Milli-Q water	10 mL		Room temperature
   Total	10 mL




8. Diluted BSA solution

   Dilute 20 mg/mL BSA solution to 0.0625, 0.125, 0.25, 0.5, 1.0, and 1.5 μg/μL in a 1.5 mL microcentrifuge tube; transfer BSA using a 50–200 μL Gilson PIPETMAN. We recommend that dissolution be performed using a vortex system (e.g., Vortex-Genie 2) at room temperature. The BSA solution should be made and used immediately with every experiment. BSA solution should be aliquoted (approximately 100 μL) and preserved at -80 °C if it will be reused. However, we recommend preparing and using a fresh BSA solution per experiment.

   Note: Prepare from 20 mg/mL BSA solution (Recipe 7).

   Volume of reagents	Volume of Solution D	Total volume	Final concentration
   150 μL of 2.0 μg/μL BSA solution	50 μL	200 μL	1.5 μg/μL
   100 μL of 2.0 μg/μL BSA solution	100 μL	200 μL	1.0 μg/μL
   100 μL of 1.0 μg/μL BSA solution	100 μL	200 μL	0.5 μg/μL
   100 μL of 0.5 μg/μL BSA solution	100 μL	200 μL	0.25 μg/μL
   100 μL of 0.25 μg/μL BSA solution	100 μL	200 μL	0.125 μg/μL
   100 μL of 0.125 μg/μL BSA solution	100 μL	200 μL	0.0625 μg/μL




9. Diluted GA-AGEs-BSA solution

   Dilute 10 mg/mL GA-AGEs-BSA in PBS(-) within a 1.5 mL microcentrifuge tube; transfer the solution using a 50–200 μL or 1–10 μL Gilson PIPETMAN.

   Note: We recommend the diluted GA-AGEs-BSA solution to be prepared before being used.

   Volume of reagents	Volume of PBS(-)	Total volume	Final concentration
   2.0 μL of 10 mg/mL GA-AGEs-BSA	98 μL	100 μL	200 ng/μL
   20 μL of 200 ng/μL GA-AGEs-BSA	60 μL	80 μL	50 ng/μL
   60 μL of 50 ng/μL GA-AGEs-BSA	40 μL	100 μL	30 ng/μL
   30 μL of 50 ng/μL GA-AGEs-BSA	70 μL	100 μL	15 ng/μL
   10 μL of 50 ng/μL GA-AGEs-BSA	90 μL	100 μL	5 ng/μL
   10 μL of 15 ng/μL GA-AGEs-BSA	90 μL	100 μL	1.5 ng/μL
   10 μL of 5 ng/μL GA-AGEs-BSA	90 μL	100 μL	0.5 ng/μL




10. HRP-conjugated molecular weight marker solution

    Dilute the HRP-conjugated molecular weight marker in PBS(-) within a 1.5 mL microcentrifuge tube; transfer the solution using the 200–1,000 μL or 1–10 μL Gilson PIPETMAN.

    Note: We recommend dissolution to be performed using a vortex system (e.g., Vortex-Genie 2) at room temperature and the diluted GA-AGEs-BSA solution to be prepared at the time of use.

    Reagent	Volume	Recommended storage condition
    HRP-conjugated molecular weight marker	3 μL	-30 °C
    PBS(-)	197 μL	Room temperature
    Total	200 μL




11. PBS-T

    Dissolve Tween-20 in PBS(-). Transfer Tween-20 using the 200–1,000 μL Gilson PIPETMAN.

    Reagent	Volume	Recommended storage condition
    Tween-20	0.5 mL	Room temperature
    PBS(-)	1,000 mL	Room temperature
    Total	1,000.5 mL




12. 5% SM-PBS-T for immunoassay

    Dissolve skim milk for immunoassay in PBS-T within a 50 mL polypropylene centrifuge tube; transfer PBS-T using the 10 mL serological pipette.

    Note: We recommend that dissolution be performed using a vortex system (e.g., Vortex-Genie 2) at room temperature and that 5% SM-PBS-T be prepared before use. However, the solution may be preserved at 4 °C for two days.

    Reagent	Quantity/Volume	Recommended storage condition
    Skim milk for immunoassay	2.5 g	4 °C
    PBS-T	50 mL	Room temperature
    Total	50 mL




13. 0.5% SM-PBS-T

    Dilute 5% SM-PBS-T to 0.5% SM-PBS-T in a 50 mL polypropylene centrifuge tube; transfer PBS-T using the 10 mL serological pipette.

    Note: We recommend that dissolution be performed using a vortex system (e.g., Vortex-Genie 2) at room temperature and that 5% SM-PBS-T be prepared before use, which may occur at room temperature. However, the solution may be preserved at 4 °C for two days.

    Reagent	Volume	Recommended storage condition
    5% SM-PBS-T	5 mL	Room temperature
    PBS-T	45 mL	Room temperature
    Total	50 mL




14. Primary antibody solution

    Mix 5 mL of 0.5% SM-PBS-T (Recipe 13) using a 10 mL serological pipette with 5 μL of polyclonal anti-GA-AGE antibody (using a 2–20 μL Gilson PIPETMAN) within a 15 mL polypropylene centrifuge tube.

    Note: We recommend that dissolution be performed using a vortex system (e.g., Vortex-Genie 2) for 10 s at room temperature. The polyclonal anti-GA-AGE antibody, which was preserved at -80 °C, should first be defrosted at 0 °C (in ice) and may then be preserved at 4 °C for half a year.

    Reagent	Volume	Recommended storage condition	Dilution ratio
    Polyclonal anti-GA-AGEs antibody	5 μL	-80 °C	1:1,000
    0.5% SM-PBS-T	5.0 mL (5,000 μL)	Room temperature
    Total (optional)	5.005 mL (5,005 μL)




15. Neutralized primary antibody solution

    Mix 5 μL of polyclonal anti-GA-AGEs antibody (2–20 μL Gilson PIPETMAN) with 125 μL of 10 mg/mL GA-AGEs-BSA (50–200 μL Gilson PIPETMAN) and 4.875 mL of 0.5% SM-PBS-T (10 mL serological pipette) within a 15 mL polypropylene centrifuge tube.

    Note: We recommend that dissolution be performed using a vortex system (e.g., Vortex-Genie 2) for 10 s at room temperature. Polyclonal anti-GA-AGEs and GA-AGEs-BSA were preserved at -80 °C and -30 °C, respectively, and defrosted on ice. We recommend that 10 mg/mL GA-AGEs-BSA be divided into 130 μL aliquots and preserved at -30 °C in advance. Moreover, we recommended the divided and preserved GA-AGEs-BSA to be used, and one of them should be for one experiment (they should not be refrozen and reused).

    Reagent	Volume	Recommended storage condition	Dilution ratio/Final concentration
    Polyclonal anti-GA-AGEs antibody	5 μL	-80 °C	1:1,000
    10 mg/mL GA-AGEs-BSA	125 μL	-30 °C	1/40 (250 μg/mL)
    0.5% SM-PBS-T	4.875 mL (4,875 μL)	Room temperature
    Total	5.005 mL (5,005 μL)




16. Secondary antibody solution

    Mix 5 mL of 0.5% SM-PBS-T (10 mL serological pipette) with 2.5 μL of polyclonal goat anti-rabbit immunological HRP-conjugated antibody (1–10 μL Gilson PIPETMAN) within a 15 mL polypropylene centrifuge tube. We recommend that dissolution be performed using a vortex system (e.g., Vortex-Genie 2) for 10 s at room temperature.

    Reagent	Volume	Recommended storage condition	Dilution ratio
    Polyclonal goat anti-rabbit immunological HRP-conjugated antibody	2.5 μL	4 °C	1:2,000
    0.5% SM-PBS-T	5 mL (5000 μL)	Room temperature
    Total	5.0025 mL (5002.5 μL)

Laboratory supplies

1. 60 mm dish (BM Equipment, catalog number: 93060)

2. 1.5 mL microcentrifuge tube (Thermo Fisher Scientific, catalog number: 3451)

3. 2.0 mL microcentrifuge tube (Watson, catalog number: 132-6201)

4. 50 mL polypropylene centrifuge tube (TrueLine; Nippon Genetics, catalog number: TR2004)

5. 15 mL polypropylene centrifuge tube (TrueLine; Nippon Genetics, catalog number: TR2000)

6. 10 mL serological pipette (BM Equipment, catalog number: 207500-SLP-10)

7. Rubber bulb for 10 mL serological pipette (AS ONE, catalog number: 6-356-4)

8. Disposable polypropylene tray (AS ONE, catalog number: 1-3145-03)

9. Dispenser (TPP, BM Equipment, catalog number: 99010)

10. 1–10 μL pre-sterilized tip (10 μL) (BM Equipment, catalog number: W10-RS)

11. 50–200 μL pre-sterilized tip (200 μL) (Watson, catalog number: 62-0887-34)

12. 200–1,000 μL pre-sterilized tip (1,000 μL) (Watson, catalog number: 38688239)

13. 96-well microplates (Becton Dickinson, catalog number: REF353072)

14. 25 mL reagent reservoir (BM Equipment, catalog number: BM-0850-1)

15. PVDF membrane (pore size: 0.45 μm) (Merck Millipore, catalog number: IPVH00010)

16. Filter paper (9 cm × 12 cm) (Bio-Rad Laboratories, catalog number: 1620161)

17. Hybri-Bag (hard type) (Cosmo Bio, catalog number: S1001)

18. Gilson PIPETMAN (GILSON, model type: 100–1,000 μL)

19. Gilson PIPETMAN (GILSON, model type: 50–200 μL)

20. Gilson PIPETMAN (GILSON, model type: 2–20 μL)

21. Gilson PIPETMAN (GILSON, model type: 1–10 μL)

22. Nichipet 7000 range: 50–200 μL (Nichiryo, Tokyo, Japan)

Equipment

1. Ultrapure Water System (Advance Toyo, model: RFU554CA)

2. Flask-trap aspirator (1,000 mL) (Biosan, Riza, Latvia; model number: FTA-1)

3. Centrifuge (Eppendorf, model: 5415R)

4. Microplate reader (Bio-Rad, model: iMark)

5. Vortex-Genie 2 (M&S Instruments, catalog number: 33230217)

6. Imager (M&S Instruments, model: Fusion FX)

7. AS-200 Heat sealer (AS ONE, catalog number: H221049/01167C)

8. Seesaw shaker (BIO CRAFT, Tokyo, model: BC-700)

9. Aspirator with water pump (AS ONE, catalog number: 1-689-02)

10. Bio-Dot SF microfiltration apparatus (48 lanes) (Bio-Rad; Figure 1)

    
    

    
    Figure 1. Bio-Dot SF microfiltration apparatus (48 lanes) containing sample template and equipped with sealing screws, sealing gasket, gasket support plate, vacuum manifold, and tubing with flow valve

Software and datasets

1. Excel software (Microsoft, Redmond, WA, USA. version 2010, 2013, 2016)

   Note: The software was installed on a standard personal computer (OS: Windows 7, 10).

2. FUSION FX software (M&S Instruments, version: 17.03)

   Note: The software was installed on a standard personal computer (OS: Windows 7) with Fusion FX imager (M&S Instruments) and was used since November 2017 at Kanazawa Medical University [11,13–16,22]. However, other chemiluminescence imagers and software may be used [10,12,17–20].

3. Stat FX software (Artech, Osaka, Japan, version: 6)

   Note: When the slot blot analysis was performed in more than three independent experiments, Statflex (version 6) was used to perform statistical analysis [10–16,18–20].

Procedure