Materials and Reagents

1.5 mL Eppendorf tubes (Eppendorf, catalog number: 022363204)
Immuno-Blot PVDF membrane (Bio-Rad, catalog number: 1620177)
Tris-HCl (pH 6.8) (Sigma-Aldrich, catalog number: T5941)
Sodium dodecyl sulfate (Sigma-Aldrich, catalog number: L3771)
Glycerol (Sigma-Aldrich, catalog number: G5516)
β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148)
EDTA (Sigma-Aldrich, catalog number: E9884)
Bromophenol blue (Sigma-Aldrich, catalog number: B8026)
NuPAGE^TM LDS sample buffer (4×) (Thermo Fisher Scientific, catalog number: NP0007)
NuPAGE^TM 3%–8%, Novex^TM tris-acetate 1.5 mm mini protein gel, 10 well (Thermo Fisher Scientific, catalog number: EA0378BOX)
NuPAGE^TM tris-acetate SDS running buffer (20×) (Thermo Fisher Scientific, catalog number: LA0041)
IRS proteins were prepared as described previously (Cao et al., 2017; Peng et al., 2022). To determine the effect of IRS acetylation on its binding to IRb, IRS1 and IRS2 proteins were acetylated by acetyltransferase P300 protein. In the acetylation assay, 2 μg of IRS1 or IRS2 were added to the reaction containing 50 mM Tris-HCl (pH 8.0), 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 1 mM dithiothreitol (DTT), 1 mM PMSF, 10 mM sodium butyrate, 0.2 μg of acetyl-CoA, and 0.2 μg of P300 (Active Motif). Samples were incubated at 30 °C for 1 h. In another acetylation assay set, acetyl-CoA was not added but it served as a positive control.
Recombinant human IRβ protein (Creative Biomart, catalog number: INSR-5093H)
Anti-IRβ (Cell Signaling Technology, catalog number: 3020); dilution: 1:500
Protein ladder (Thermo Fisher Scientific, catalog number: 26634)
Tris (Sigma-Aldrich, catalog number: T1503)
Glycine (Sigma-Aldrich, catalog number: G8898)
Methanol (Sigma-Aldrich, catalog number: 34860)
NaCl (Sigma-Aldrich, catalog number: 9888)
Tween-20 (Sigma-Aldrich, catalog number: P1379)
Skim milk powder (Bio-Rad, catalog number: 1706404XTU)
DTT (Sigma-Aldrich, catalog number: D0632)
KH₂PO₄(Sigma-Aldrich, catalog number: P5655)
Na₂HPO₄(Sigma-Aldrich, catalog number: S9763)
Guanidine-HCl (Sigma-Aldrich, catalog number: G3272)
ECL kit (Pierce^TMECL western blotting substrate) (Thermo Fisher Scientific, catalog number: 32106)
Loading buffer (see Recipes)
Wet transfer buffer (see Recipes)
Denaturing and renaturing buffers (see Table 1) (see Recipes)
Protein-binding buffer (see Recipes)
PBST buffer (see Recipes)

Equipment

Mini gel tank (Thermo Fisher Scientific, catalog number: A25977)
PowerPac^TM basic power supply (Bio-Rad, catalog number: 1645050)
ChemiDoc XRS+ gel imaging system (Bio-Rad)
Mini trans-blot electrophoretic transfer cell (Bio-Rad, catalog number: 1703930)

Procedure

Denaturing the protein samples
1. Prepare 4× loading buffer.
2. Mix the loading buffer (4×) and NuPAGE^TM LDS sample buffer (4×) in 1:1 volume.
3. Apply the mix to the purified protein of mouse IRS1 (0.2–0.4 μg/μL) and IRS2 (0.2–0.5 μg/μL) (final 1×) in 1.5 mL Eppendorf tubes, at 95 °C for 10 min, and put on ice right after.
Notes:
1. The mixture of loading buffer and NuPAGE^TM LDS sample buffer (final 1×) can be used as a negative control.
2. During the heating process, pay close attention to prevent the sample from volatilizing due to the tubes’ caps opening. Before loading the sample, collect the water vapor on the cap by completing a short spin.

Separating protein samples by electrophoresis
Load the denatured IRS1 and IRS2 protein samples onto the Novex^TM mini protein gel along with the protein ladder. Run at 80 V in the stacking gel and then turn to 120–150 V in the resolving gel, until your target protein migrates to the middle of the resolving gel.
Notes:
1. Sample volume should not be >50 μL for a 1.5 mm 10-well gel. In our experiment, we load 5–10 μg of IRS1 (180 kD) protein and 5–12 μg of IRS2 (185 kD) protein.
2. When the protein samples run into the resolving gel, transfer the electrophoresis tank into the refrigerator set at 4 °C.
3. Based on the migration of the protein ladder, the position of the target protein in the gel can be estimated.

Transferring the protein to the membrane
1. Membrane preparation: pre wet the PVDF membrane first in methanol for 20 s, then place in ultrapure water for one quick rinse, followed by sitting in ultrapure water for 5 min, and finally in the wet transfer buffer (for at least 15 min).
2. After electrophoresis is complete, wash the electrophoresis plates with pure water, place the gel over the blotting paper, carefully place the pre wet PVDF membrane onto the gel, place a second blotting paper onto the PVDF membrane, and place a sponge support pad.
3. In this assay, we use wet transfer [100 V/gel, 2 h (for 1.5 mm gel), room temperature (RT)] or 10 mA/gel, overnight at 4 °C.
  Caution:
  1. To open and remove gel(s) from the pre-cast gel cassettes, please refer to the “NuPAGE Technical Guide” at https://www.thermofisher.cn/order/catalog/product/EA0375PK2.
  2. Make sure that there are no air bubbles present in the transfer sandwich.

Denaturing/renaturing of proteins on the membrane

After the transfer is complete, take the membrane out of the sandwich and rinse with ultrapure water twice.

Incubate the membrane in the denaturing and renaturing buffers (freshly prepared) from high to low concentrations of guanidine-HCl as shown in Table 1. Finally, incubate the membrane in the denaturing and renaturing buffer without guanidine-HCl overnight at 4 °C.

Table 1. Denaturing and renaturing buffers with varying concentrations of guanidine-HCl

Guanidine-HCl concentration	6 M	3 M	1 M	0.1 M	0 M
80% glycerol (mL)	3.125	3.125	3.125	3.125	3.125
5 M NaCl (mL)	0.5	0.5	0.5	0.5	0.5
1 M Tris pH 7.5 (mL)	0.5	0.5	0.5	0.5	0.5
0.5 M EDTA (mL)	0.05	0.05	0.05	0.05	0.05
10% tween-20 (mL)	0.25	0.25	0.25	0.25	0.25
8 M guanidine-HCl (mL)	18.75	9.3	3.13	0.31	0
Milk powder (g)	0.5	0.5	0.5	0.5	0.5
1M DTT (μL)	25	25	25	25	25
ddH₂O (mL)	1.825	12.195	17.445	20.265	20.575
Total volume (mL)	25	25	25	25	25
Time/temperature	30 min/RT	30 min/RT	30 min/RT	30 min/4 °C	Overnight/4 °C

Caution: Completely renaturing proteins on the membrane is critical for subsequent protein–protein binding assays.

Blocking the membrane with 5% milk in PBST buffer for 1 h, at RT

Incubating the membrane with recombinant human IRβ protein
1. After blocking, wash the membrane with PBST buffer three times for 5 min.
2. Apply 3–10 μg of IRβ in PBST buffer containing 3% milk and incubate with the membrane at 4 °C overnight.

Detecting the IRβ bound to IRS1/2 on the membrane
1. Wash off un-bound IRβ with PBST buffer three times for 10 min.
2. Incubate with anti-IRβ (1:250 dilution) in 3% milk PBST, at 4 °C overnight.
3. Wash the membrane with PBST buffer, three times for 10 min.
4. Incubate the membrane with the secondary antibody (1:5,000 dilution) for 1 h at RT.
5. Wash the membrane with PBST buffer, three times for 10 min.
6. Rinse the membrane with PBS for 5 min.

Developing the membrane with the chemiluminescent ECL kit according to the instructions
Briefly, prepare the chemiluminescent substrate reagent by mixing the detection reagent 1 and 2 at 1:1 volume. Incubate the blot with the chemiluminescent substrate for 2 min. Remove the blot from the substrate and place it in the ChemiDoc XRS+ gel imaging system to get image.
Caution: Use a sufficient volume to ensure that the blot is completely wet with the chemiluminescent substrate and does not become dry (0.1 mL/cm²).

Recipes

Loading buffer
50 mM Tris-HCl (pH 6.8)
2% sodium dodecyl sulfate
10% glycerol
1% β-mercaptoethanol
12.5 mM EDTA
0.02% bromophenol blue
This loading dye can be stored at RT (-20 °C) for up to six months.

Wet transfer buffer
25 mM Tris
192 mM glycine
20% methanol
The buffer should be freshly prepared.

Denaturing and renaturing buffers
100 mM NaCl
20 mM Tris (pH 7.6)
10% glycerol
0.1% Tween-20
2% skim milk powder
1 mM DTT
This should be freshly prepared.

Protein-binding buffer
100 mM NaCl
20 mM Tris (pH 7.6)
0.5 mM EDTA
10% glycerol
0.1% Tween-20
2% skim milk powder
1 mM DTT
The solution should be freshly prepared.

PBST buffer
4 mM KH₂PO₄
16 mM Na₂HPO₄
115 mM NaCl (pH 7.4)
0.05% Tween-20
Prepare 10× PBST and store at RT for up to three months. Dilute to 1× before use.

Acknowledgments

This work was supported in part by grants from the NIH: R01DK107641 and R01DK120309. This protocol was modified from a previous publication (Wu et al., 2007).

Competing interests

We declare no competing interest.

References

Cao, J., Peng, J., An, H., He, Q., Boronina, T., Guo, S., White, M. F., Cole, P. A. and He, L. (2017). Endotoxemia-mediated activation of acetyltransferase P300 impairs insulin signaling in obesity. Nat Commun 8(1): 131.
Cheatham, B. and Kahn, C. R. (1995). Insulin action and the insulin signaling network. Endocr Rev 16(2): 117-142.
Kaido, M., Inoue, Y., Takeda, Y., Sugiyama, K., Takeda, A., Mori, M., Tamai, A., Meshi, T., Okuno, T. and Mise, K. (2007). Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana. Mol Plant Microbe Interact 20(6): 671-681.
Machado-Neto, J. A., Fenerich, B. A., Rodrigues Alves, A. P. N., Fernandes, J. C., Scopim-Ribeiro, R., Coelho-Silva, J. L. and Traina, F. (2018). Insulin Substrate Receptor (IRS) proteins in normal and malignant hematopoiesis. Clinics 73(suppl 1): e566s.
Peng, J. and He, L. (2018). IRS posttranslational modifications in regulating insulin signaling. J Mol Endocrinol 60(1): R1-R8.
Peng, J., Ramatchandirin, B., Wang, Y., Pearah, A., Namachivayam, K., Wolf, R. M., Steele, K., MohanKumar, K., Yu, L., Guo, S., White, M. F., Maheshwari, A. and He, L. (2022). The P300 acetyltransferase inhibitor C646 promotes membrane translocation of insulin receptor protein substrate and interaction with the insulin receptor. J Biol Chem 298(3): 101621.
Sweet, L. J., Morrison, B. D. and Pessin, J. E. (1987). Isolation of functional alpha beta heterodimers from the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex. A structural basis for insulin binding heterogeneity. J Biol Chem 262(15): 6939-6942.
White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A. and Kahn, C. R. (1988). Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity. Cell 54(5): 641-649.
Wu, Y., Li, Q. and Chen, X. Z. (2007). Detecting protein-protein interactions by Far western blotting. Nat Protoc 2(12): 3278-3284.
Yip, C. C. and Ottensmeyer, P. (2003). Three-dimensional structural interactions of insulin and its receptor. J Biol Chem 278(30): 27329-27332.