Slot Blot Analysis of Intracellular Glyceraldehyde-Derived Advanced Glycation End Products Using a Novel Lysis Buffer and Polyvinylidene Difluoride Membrane

Advanced glycation end products (AGEs) are formed through the reaction/modification of proteins by saccharides (e.g., glucose and fructose) and their intermediate/non-enzymatic products [e.g., methylglyoxal and glyceraldehyde (GA)]. In 2017, Dr. Takanobu Takata et al. developed the novel slot blot method to quantify intracellular GA-derived AGEs (GA-AGEs). Although the original method required nitrocellulose membranes, we hypothesized that the modified proteins contained in the AGEs may be effectively probed on polyvinylidene difluoride (PVDF) membranes. Because commercial lysis buffers are unsuitable for this purpose, Dr. Takata developed the slot blot method using an in-house-prepared lysis buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) that effectively probes AGEs onto PVDF membranes. The slot blot method also entails the calculation of Tris, urea, thiourea, and CHAPS concentrations, as well as protein and mass to be probed onto the PVDF membranes. GA-AGE-modified bovine serum albumin (BSA, GA-AGEs-BSA) is used to draw a standard curve and perform neutralization against a non-specific combination of anti-GA-AGEs antibodies, thereby enabling the quantification of GA-AGEs in cell lysates. This paper presents the detailed protocol for slot blot analysis of intracellular GA-AGE levels in C2C12 cells. Key features • This protocol leverages the idea that advanced glycation end products are modified proteins. • The lysis buffer containing Tris, urea, thiourea, and CHAPS enables probing proteins onto PVDF membranes. • Intracellular GA-AGE levels may be quantified for some cell types using polyclonal anti-GA-AGE antibodies and standard GA-AGE-modified BSA. • The lysis buffer may be simultaneously prepared with the cell lysate. • There is no limit to the type of cultured cells used in the preparation of cell lysate.

This protocol is used in: Diabetol.Metab.Syndr.(2020), DOI: 10.1186/s13098-020-00561-z Advanced glycation end products (AGEs) are formed through the reaction/modification of proteins by saccharides (e.g., glucose and fructose) and their intermediate/non-enzymatic products [e.g., methylglyoxal and glyceraldehyde (GA)].In 2017, Dr. Takanobu Takata et al. developed the novel slot blot method to quantify intracellular GA-derived AGEs (GA-AGEs).Although the original method required nitrocellulose membranes, we hypothesized that the modified proteins contained in the AGEs may be effectively probed on polyvinylidene difluoride (PVDF) membranes.Because commercial lysis buffers are unsuitable for this purpose, Dr. Takata developed the slot blot method using an in-house-prepared lysis buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) that effectively probes AGEs onto PVDF membranes.The slot blot method also entails the calculation of Tris, urea, thiourea, and CHAPS concentrations, as well as protein and mass to be probed onto the PVDF membranes.GA-AGE-modified bovine serum albumin (BSA, GA-AGEs-BSA) is used to draw a standard curve and perform neutralization against a nonspecific combination of anti-GA-AGEs antibodies, thereby enabling the quantification of GA-AGEs in cell lysates.This paper presents the detailed protocol for slot blot analysis of intracellular GA-AGE levels in C2C12 cells.

Background
The quantification of intracellular advanced glycation end products (AGEs) is particularly useful in the fields of biochemistry, molecular biology, and protein engineering [1].AGEs are a type of modified proteins that can be extracted from cultured cells and tissues [2] and indirectly measured using the enzyme-linked immunosorbent assay (ELISA) [3,4].However, some researchers prefer using a slot blot approach [5][6][7], because identifying or quantifying some types of AGEs may be difficult using ELISA.Previous slot blot methods for the quantification of AGEs were limited by their use of nitrocellulose membranes and radioimmunoprecipitation (RIPA) buffer [5,6].Although nitrocellulose membranes are useful in column chromatography, polyvinylidene difluoride (PVDF) membranes are more durable and show greater protein adsorption ability [8,9].However, there is a lack of suitable lysis buffers for probing proteins onto PVDF membranes.Although RIPA buffer is used in western blotting, it is less suitable in slot blot analyses with PVDF membranes [8,9].Consequently, Dr. Takanobu Takata developed a lysis buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) to quantify intracellular AGEs using the slot blot analysis [8].The improved probing efficacy of this lysis buffer may be related to protein carbamoylation [8,9] and the absence of Triton-X, which can inhibit the probing of proteins onto PVDF membranes [8,9].In 2017, the novel slot blot method was used to accurately quantify intracellular glyceraldehyde (GA)-derived AGEs (GA-AGEs) [10].From 2017 to 2022, Takata et al. applied this method in the quantification of intracellular GA-AGEs in cells and tissue lysates of the pancreas [10,11], heart [12,13], skeletal muscles [14], liver [15][16][17][18][19], and bone [20] using (i) the novel lysis buffer, (ii) standard GA-AGEs modified bovine serum albumin (BSA), and (iii) neutralization using anti-GA-AGEs antibodies.In this method, the standard GA-AGEs-BSA is used to estimate standard curves [10]; neutralization using a non-specific combination of polyclonal anti-GA-AGEs antibodies [21] avoids biases in the quantification

Medium for cell incubation
Mix the reagents on a laminar flow hood.

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

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.

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).8

A. Incubation of C2C12 cell line and treatment with glyceraldehyde
Note: Here, we only provide the simplified procedure for the slot blot analysis.A detailed description may be found in the Materials and Methods in Takata et al. [14] (DOI: 10.1186/s13098-020-00561-z).
1. Seed 1.9 × 10 4 cells/cm 2 onto a 60 mm dish and incubate in a CO2 incubator for 24 h on DMEM supplemented with penicillin/streptomycin and fetal bovine serum.2. After changing the medium, treat the cells with 0, 0.5, 1, 1.5, and 2 mM glyceraldehyde and incubate in the CO2 incubator for 24 h.Note: The medium should be refreshed 24 h after seeding.

B. Removing medium and washing cells with PBS(-)
1. Remove culture medium by decantation and aspirate the residual medium with a 1,000 mL trap-flask aspirator.Perform this step twice.Note: After decantation, ~300 μL of the culture medium should remain.2. Add 7.0 mL of PBS(-), decant, and aspirate the residual PBS(-) with the flask-trap aspirator.Perform this step twice.

C. Preparation of cell lysates
1. Add 300 μL of Solution D into a 60 mm dish and scrape and move the cells into a 1.5 mL microcentrifuge tube.2. Add cells to Solution D and harvest with a dispenser.
Note: Bradford dye reagent should be preserved at 4 °C.However, we recommend it to be kept at 22-28 °C for 30-60 min before being added into BSA in Solution D and the cell lysates.4. Transfer 200 μL of Bradford dye reagent using the Nichipet 7000 to the BSA in Solution D and cell lysate in the 96-well microplates.5.After 5 and 10 min, measure absorbance (595 nm) using the iMark microplate reader.

E. PVDF membrane and filter papers incubated in methanol and/or PBS(-)
1. Cut a PVDF membrane into 9 cm × 12 cm sections and incubate in methanol for 1 min at room temperature.
Note: This operation should be performed in a fume hood to avoid exposure to methanol.2. Submerge the PVDF membrane sections in PBS(-) at room temperature.3. Submerge also three filter papers (9 cm × 12 cm) in PBS(-) at room temperature.4. Incubate both the PVDF membrane sections and three filter papers in the PBS(-) for 1 h at room temperature.
Note: Because the PVDF membrane may be hydrophobic after incubation in methanol, excess methanol should be removed using the three filter papers, and the membrane sections should be sufficiently submerged in the PBS(-).

F. PVDF membrane and filter papers prepared for slot blot apparatus
1. Set the sealing gasket onto the vacuum manifold (Figure 1).11 Published: Jul 20, 2024 2. Set the gasket support plate onto the vacuum manifold (Figure 1).
3. Set each filter paper (a total of three) onto the gasket support plate (Figures 1, 2). 4. Set the PVDF membrane onto the filter papers (Figure 2). 5. Incubate both the PVDF membrane and filter papers in PBS(-) for 1 h at room temperature.
6. Fix the sample template with attached sealing screw to the sealing gasket and tighten the four screws (Figures 1-3).
Note: Any air between the PVDF membranes and filter papers should be removed.Do not dry the PVDF membrane before adding the PBS(-) in section G.If section G cannot be performed before the PVDF membrane is dried, a little PBS(-) may be added to it.Because the PVDF membrane may be hydrophobic after incubation in methanol, excess methanol should be removed using the three filter papers, and the membrane sections should be sufficiently submerged in the PBS(-).Ensure that the four screws are tightened appropriately.L. Sample, GA-AGEs-BSA, and HRP-conjugated marker solution applied onto the PVDF membrane and removed with water aspiration 1. Apply 200 μL of sample, GA-AGEs-BSA, and HRP-conjugated marker solutions.Note: The 50-200 μL Gilson PIPETMAN should preferably be used.This operation should be performed with the water aspirator (Figure 5). 2. Place the slot blot apparatus on a stand near the water aspirator.
Note: Any stand may be used for the experiment, as long as it has a height of 25-30 cm (Figure 6). 3. Connect a tube from the flow valve to the water aspirator (Figure 6). 4. Vacuum the sample, GA-AGEs-BSA, and HRP-conjugated marker solutions with water aspiration while opening one valve for air (Figure 7A). 5.For complete sample addition, we recommend vacuuming with water aspiration while the valve is closed for air (3-5 s) (Figure 7B).Note: Although water aspiration pressure is not specified, this can be estimated.Vacuuming with water aspiration was performed at the Kanazawa Medical University, where the water supply is collected in a water tank and redistributed between laboratories.Here, the water pressure is consistent with that of a typical household (0.15-0.74 MPa) or corporate water supply system in Uchinada (0.20-0.23 MPa), according to data from the Ministry of Health, Labour, and Welfare in Japan.Therefore, the water supply system at Kanazawa Medical University has been adjusted to a pressure of 0.20-0.23MPa.

3 .
Incubate cells in Solution D on ice for 20 min, during which the cell suspension is subjected to five pipetting operations, repeated three times.Note: This operation is performed with 5-6 min intervals.4. Centrifuge cells at 10,000× g for 15 min at 4 °C with the 5415R centrifuge. 5. Collect the supernatants in the 1.5 mL microcentrifuge tube.Note: Cell lysates are preserved at -80 °C.Both cell lysates and Solution D are preserved at -80 °C until the protein concentrations are measured, because the components of Solution D should remain in the same conditions.

Figure 2 .
Figure 2. PVDF membranes and filter papers set onto slot blot apparatus.The white closed square represents the PVDF membrane, the gray closed squares represent the filter papers, and the blue closed square represents the sealing gasket.

Figure 3 .G.Figure 4 . 1 .
Figure 3. Slot blot apparatus with 48 lanes for PVDF membrane with sample template attached using sealing screws

Figure 5 .Figure 6 .Figure 7 .
Figure 5. Water aspirator (AS ONE) in the laboratory at Kanazawa Medical University

Cite as: Takata, T. et al. (2024). Slot Blot Analysis of Intracellular Glyceraldehyde-Derived Advanced Glycation End Products Using a Novel Lysis Buffer and Polyvinylidene Difluoride Membrane. Bio-protocol 14(14): e5038. DOI: 10.21769/BioProtoc.5038. 5 Published: Jul 20, 2024 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.
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.6.Solution DPrepare 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.

10. HRP-conjugated molecular weight marker solution Dilute
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.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.
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.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 Cite as: Takata, T. et al. (2024).Slot Blot Analysis of Intracellular Glyceraldehyde-Derived Advanced Glycation End Products Using a Novel Lysis Buffer and Polyvinylidene Difluoride Membrane.Bio-protocol 14(14): e5038.DOI: 10.21769/BioProtoc.5038.6Published:Jul20,2024 be prepared.We believe that BSA fraction IV is suitable for the Bradford method in our protocol.9.Diluted GA-AGEs-BSA solutionDilute 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.Cite as: Takata, T. et al. (2024).Slot Blot Analysis of Intracellular Glyceraldehyde-Derived Advanced Glycation End Products Using a Novel Lysis Buffer and Polyvinylidene Difluoride Membrane.Bio-protocol 14(14): e5038.DOI: 10.21769/BioProtoc.5038.