Figure 1: Insulin Target Protein Structure.

Insulin Introduction

Protein Function

Insulin decreases blood glucose concentration, increases cell permeability to monosaccharides, amino acids, and fatty acids, and accelerates glycolysis, the pentose phosphate cycle, and glycogen synthesis in the liver.

Protein Expression

• Expressed in type B pancreatic cells.

Protein Characteristics

Islet cells are relatively low in proportion within the pancreas. When detecting insulin in pancreatic tissue, staining is observed only in specific cells.

Figure 2: Insulin IHC image, Anti-Insulin antibody [EPR17359] (ab181547).

Protein Localization

• Secreted.

Figure 3: Insulin ICCimage, Anti-Insulin antibody [EPR17359] (ab181547).

Isoforms & Post-Translational Modifications

• Human (P01308): Isoform 1: 12 kDa, Isoform 2: 21 kDa (predicted)
• Mouse (P01325): 12 kDa (predicted)
• Rat (P01322): 12 kDa (predicted)
• Post-Translational Modifications: disulfide bond.

WB Experiment Tips

Precautions

• Insulin exhibits specificity in expression, primarily in pancreatic islet beta cells. However, due to the relatively low proportion of islet cells in the pancreas, detecting insulin in pancreatic tissue lysates may result in weak signals. We recommend increasing the sample loading amount or reducing antibody dilution to enhance detection signals.
• The human pancreas is larger than those of mice and rats. Therefore, the proportion of islet cells in human pancreatic tissue lysates is lower, making detecting insulin signals in WB applications more challenging.
• Insulin protein has a predicted molecular weight of 12 kDa. Experimentation should be conducted using protocols designed for low molecular weight proteins. Key control points provide specific details.
• If there are significant background bands or aggregates, consider trying non-boiled samples.

Positive controls:

• Mouse and rat pancreas
• Beta-Tc-6 (Mouse pancreas insulinoma beta cell) whole cell lysate

Example results:

Figure 4: WB-Anti-Insulin antibody [EPR17359] (ab181547).

Lane 1: 20 µg human pancreas
Lane 2: 20 µg mouse pancreas
Lane 3: 20 µg rat pancreas

Predicted band size: 12 kDa
Notes: This antibody detects samples from mouse and rat but cannot detect human samples.

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Keep samples on ice throughout the entire sample preparation process.
3. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. For target proteins with smaller molecular weights (e.g., <25 kDa), it is recommended to use a 15% separating gel for electrophoresis.
2. Load at least 20μg of total protein from cell lysate or tissue homogenate.

Transfer:

1. For target proteins with a lower molecular weight, it is recommended to use a PVDF membrane with a pore size of 0.22 μm.
2. We recommend using 20% methanol in the transfer buffer for target proteins with a lower molecular weight.
3. We recommend staining the membrane with Ponceau S after the transfer to confirm its success.

References

1. Květoslava Křížková, Václav Veverka, Lenka Maletínská, Rozálie Hexnerová, Andrzej M Brzozowski, Jiří Jiráček, Lenka Žáková. Structural and functional study of the GlnB22-insulin mutant responsible for maturity-onset diabetes of the young. PLoS One. 2014 Nov 25;9(11):e112883. doi:10.1371/journal.pone.0112883. eCollection 2014.
2. M A Soos, C E Field, K Siddle. Purified hybrid insulin/insulin-like growth factor-I receptors bind insulin-like growth factor-I, but not insulin, with high affinity. Biochem J. 1993 Mar 1;290 ( Pt 2)(Pt 2):419-26. doi: 10.1042/bj2900419.