Choose Your Country or Region

Anti-Insulin Rabbit Antibody [K13G15]

Catalog No.: F0436

Application: Reactivity: Human, Mouse, Rat

Usage Information

Dilution
IHC1:6400 - 1:25600 IF1:200 - 1:800 FCM1:50 - 1:200

Application
IHC, IF, FCM

Reactivity
Human, Mouse, Rat

Source
Rabbit

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
12 kDa

Positive Control	Human pancreas; Mouse pancreas; Rat pancreas; β-TC-6 cell; INS-1 cell
Negative Control	C2C12 cell

Exprimental Methods

IF
Experimental Protocol： Specimen Preparation 1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS. NOTE: Paraformaldehyde is toxic, use only in a fume hood. 2. Fix cells for 15 min at room temperature. 3. Aspirate fixative, rinse three times in 1X PBS for 5 min each. 4. Proceed with Immunostaining. Immunostaining 1. Add theblocking buffer and incubate for 60 min at RT. 2. Prepare primary antibody diluent in antibody dilution buffer as recommended . 3. Aspirate blocking solution, apply diluted primary antibody. 4. Incubate overnight at 4°C. 5. Rinse three times in 1X PBS for 5 min each. 6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark. 7. Rinse three times in 1X PBS for 5 min each. 8. Mount slides usingmounting medium with DAPI and cover with coverslips. 9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 23°C protected from light.

Experimental Protocol：

Specimen Preparation

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS.

NOTE: Paraformaldehyde is toxic, use only in a fume hood.

2. Fix cells for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Proceed with Immunostaining.

Immunostaining

1. Add theblocking buffer and incubate for 60 min at RT.

2. Prepare primary antibody diluent in antibody dilution buffer as recommended .

3. Aspirate blocking solution, apply diluted primary antibody.

4. Incubate overnight at 4°C.

5. Rinse three times in 1X PBS for 5 min each.

6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.

7. Rinse three times in 1X PBS for 5 min each.

8. Mount slides usingmounting medium with DAPI and cover with coverslips.

9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 23°C protected from light.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
Insulin (K13G15) Rabbit mAb recognizes endogenous levels of total Insulin protein.

Subcellular Location
Secreted

Uniprot ID
P01308

Clone
K13G15

Synonym(s)
Insulin, INS, c-peptide

Background
Insulin is a peptide hormone composed of 51 amino acids, secreted by the β-cells of the pancreas. It plays a vital role in maintaining glucose homeostasis by binding to the insulin receptor (IR), a transmembrane receptor tyrosine kinase composed of two extracellular α-subunits responsible for ligand binding and two intracellular β-subunits that mediate signal transduction. Upon insulin engagement, the receptor undergoes autophosphorylation on its tyrosine residues, activating its kinase activity. This leads to phosphorylation of insulin receptor substrates (IRS), which in turn recruit and activate phosphoinositide 3-kinase (PI3K). Activated PI3K generates phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which facilitates the activation of AKT (protein kinase B). Activated AKT regulates several downstream metabolic processes: it promotes glucose uptake in skeletal muscle and adipose tissue by inducing the translocation of the GLUT4 glucose transporter to the cell membrane, suppresses hepatic glucose production by phosphorylating and inhibiting the transcription factor FOXO1, and enhances glycogen storage by inhibiting glycogen synthase kinase 3 (GSK3). Additionally, insulin signaling inhibits lipolysis through PDE3B and ABHD15-mediated suppression of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), while simultaneously promoting lipogenesis and protein synthesis via activation of the mTORC1 complex. Insulin resistance occurs when cells become less responsive to insulin signaling, often due to impairments at multiple levels of the pathway. These may include decreased insulin receptor numbers, defective IRS phosphorylation, or disrupted PI3K/AKT signaling. Contributing factors include chronic inflammation, oxidative stress, and accumulation of lipid intermediates such as diacylglycerols and ceramides, which interfere with IRS-1 and AKT activity. Furthermore, ectopic lipid deposition in tissues like the liver and skeletal muscle can activate stress-related kinases, including JNK and PKCθ, leading to serine phosphorylation of IRS proteins and subsequent inhibition of insulin signaling. Mitochondrial dysfunction and endoplasmic reticulum (ER) stress also contribute to impaired glucose metabolism, driving persistent hyperglycemia and progressive β-cell dysfunction—hallmarks of type 2 diabetes.

Background

Insulin is a peptide hormone composed of 51 amino acids, secreted by the β-cells of the pancreas. It plays a vital role in maintaining glucose homeostasis by binding to the insulin receptor (IR), a transmembrane receptor tyrosine kinase composed of two extracellular α-subunits responsible for ligand binding and two intracellular β-subunits that mediate signal transduction. Upon insulin engagement, the receptor undergoes autophosphorylation on its tyrosine residues, activating its kinase activity. This leads to phosphorylation of insulin receptor substrates (IRS), which in turn recruit and activate phosphoinositide 3-kinase (PI3K). Activated PI3K generates phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which facilitates the activation of AKT (protein kinase B). Activated AKT regulates several downstream metabolic processes: it promotes glucose uptake in skeletal muscle and adipose tissue by inducing the translocation of the GLUT4 glucose transporter to the cell membrane, suppresses hepatic glucose production by phosphorylating and inhibiting the transcription factor FOXO1, and enhances glycogen storage by inhibiting glycogen synthase kinase 3 (GSK3). Additionally, insulin signaling inhibits lipolysis through PDE3B and ABHD15-mediated suppression of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), while simultaneously promoting lipogenesis and protein synthesis via activation of the mTORC1 complex. Insulin resistance occurs when cells become less responsive to insulin signaling, often due to impairments at multiple levels of the pathway. These may include decreased insulin receptor numbers, defective IRS phosphorylation, or disrupted PI3K/AKT signaling. Contributing factors include chronic inflammation, oxidative stress, and accumulation of lipid intermediates such as diacylglycerols and ceramides, which interfere with IRS-1 and AKT activity. Furthermore, ectopic lipid deposition in tissues like the liver and skeletal muscle can activate stress-related kinases, including JNK and PKCθ, leading to serine phosphorylation of IRS proteins and subsequent inhibition of insulin signaling. Mitochondrial dysfunction and endoplasmic reticulum (ER) stress also contribute to impaired glucose metabolism, driving persistent hyperglycemia and progressive β-cell dysfunction—hallmarks of type 2 diabetes.

References
https://pubmed.ncbi.nlm.nih.gov/30067154/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
If you have any other enquiries, please leave a message.

* Indicates a Required Field

Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%