Somatostatin Antibody [J16B12]

Catalog No.: F3926

Application: Reactivity: Human, Mouse, Rat

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代表番号: 045-509-1970|電子メール：sales@selleck.co.jp

使用情報

Dilution
IHC1:25-1:100 IF1:25-1:100

Application
IHC, IF

Source
Mouse Monoclonal Antibody

Reactivity
Human, Mouse, Rat

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
13 kDa

プロトコール

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°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

生物学的記述

Specificity
Somatostatin Antibody [J16B12] detects endogenous levels of total Somatostatin protein.

Uniprot ID
P61278

Clone
J16B12

Synonym(s)
somatostatin , SMST

Background
Somatostatin is a cyclic peptide hormone that arises from tissue‑specific processing of a single preprosomatostatin precursor into predominantly 14‑ and 28‑residue active forms and is produced by hypothalamic neurons, pancreatic δ cells, and enteroendocrine D cells, where it functions as a broadly inhibitory regulator of endocrine, neural, and gastrointestinal activity through a dedicated family of G protein–coupled receptors termed SSTR1–SSTR5. The mature peptides share a central Phe‑Trp‑Lys‑Thr motif required for high‑affinity receptor binding and adopt a constrained cyclic conformation that positions this pharmacophore to engage ligand‑binding pockets formed by transmembrane domains III–VII and extracellular loop 2 of SSTRs, with SST‑14 preferentially acting in the brain and SST‑28 enriched in gastrointestinal actions. All five somatostatin receptors are seven‑transmembrane Gi/o‑coupled receptors that, upon ligand binding, inhibit adenylyl cyclase and reduce intracellular cAMP, activate phosphotyrosine phosphatases, and modulate MAP kinase cascades, while individual subtypes couple to additional effectors including inwardly rectifying K⁺ channels, voltage‑gated Ca²⁺ channels, Na⁺/H⁺ exchangers, and phospholipase C or A₂, allowing cell type–specific integration of the inhibitory signal with membrane excitability and secretory machinery. These signaling mechanisms converge on suppression of hormone and transmitter release by lowering cAMP and Ca²⁺ and by a distal effect on exocytosis, while receptor‑linked phosphatase–MAPK pathways mediate cell‑cycle control and survival, with SSTR1, 2, 4, and 5 inducing cytostatic responses via retinoblastoma protein and p21, and SSTR3 uniquely triggering p53‑ and Bax‑dependent apoptosis, giving somatostatin a direct antiproliferative and pro‑apoptotic profile in responsive tissues and tumors. As a hypothalamic hypophysiotropic hormone, somatostatin released into the portal circulation inhibits pituitary secretion of growth hormone and thyroid‑stimulating hormone, while peripheral somatostatin from pancreatic and gastrointestinal sources suppresses insulin, glucagon, gastrin, secretin, cholecystokinin, and other gut hormones, slows gastric emptying and intestinal motility, and reduces splanchnic blood flow, placing somatostatin at the center of feedback loops that coordinate nutrient intake with endocrine and digestive activity. In the nervous system, somatostatin acts as a neuromodulator co‑localized with GABA and other transmitters, influencing synaptic transmission, pain processing, and cognitive circuits through pre‑ and postsynaptic SSTRs and contributing to regulation of neuronal excitability and plasticity. Dysregulated somatostatin signaling contributes to endocrine and neoplastic disease: reduced hypothalamic somatostatin tone or impaired receptor function is associated with excess growth hormone secretion in acromegaly, while high SSTR expression on neuroendocrine tumors underlies their sensitivity to somatostatin analogs that exploit the same inhibitory pathways to control hormone hypersecretion and tumor growth, and overproduction of somatostatin in somatostatinomas leads to diabetes, steatorrhea, and gallstones through marked suppression of insulin and gastrointestinal secretions.

Background

Somatostatin is a cyclic peptide hormone that arises from tissue‑specific processing of a single preprosomatostatin precursor into predominantly 14‑ and 28‑residue active forms and is produced by hypothalamic neurons, pancreatic δ cells, and enteroendocrine D cells, where it functions as a broadly inhibitory regulator of endocrine, neural, and gastrointestinal activity through a dedicated family of G protein–coupled receptors termed SSTR1–SSTR5. The mature peptides share a central Phe‑Trp‑Lys‑Thr motif required for high‑affinity receptor binding and adopt a constrained cyclic conformation that positions this pharmacophore to engage ligand‑binding pockets formed by transmembrane domains III–VII and extracellular loop 2 of SSTRs, with SST‑14 preferentially acting in the brain and SST‑28 enriched in gastrointestinal actions. All five somatostatin receptors are seven‑transmembrane Gi/o‑coupled receptors that, upon ligand binding, inhibit adenylyl cyclase and reduce intracellular cAMP, activate phosphotyrosine phosphatases, and modulate MAP kinase cascades, while individual subtypes couple to additional effectors including inwardly rectifying K⁺ channels, voltage‑gated Ca²⁺ channels, Na⁺/H⁺ exchangers, and phospholipase C or A₂, allowing cell type–specific integration of the inhibitory signal with membrane excitability and secretory machinery. These signaling mechanisms converge on suppression of hormone and transmitter release by lowering cAMP and Ca²⁺ and by a distal effect on exocytosis, while receptor‑linked phosphatase–MAPK pathways mediate cell‑cycle control and survival, with SSTR1, 2, 4, and 5 inducing cytostatic responses via retinoblastoma protein and p21, and SSTR3 uniquely triggering p53‑ and Bax‑dependent apoptosis, giving somatostatin a direct antiproliferative and pro‑apoptotic profile in responsive tissues and tumors. As a hypothalamic hypophysiotropic hormone, somatostatin released into the portal circulation inhibits pituitary secretion of growth hormone and thyroid‑stimulating hormone, while peripheral somatostatin from pancreatic and gastrointestinal sources suppresses insulin, glucagon, gastrin, secretin, cholecystokinin, and other gut hormones, slows gastric emptying and intestinal motility, and reduces splanchnic blood flow, placing somatostatin at the center of feedback loops that coordinate nutrient intake with endocrine and digestive activity. In the nervous system, somatostatin acts as a neuromodulator co‑localized with GABA and other transmitters, influencing synaptic transmission, pain processing, and cognitive circuits through pre‑ and postsynaptic SSTRs and contributing to regulation of neuronal excitability and plasticity. Dysregulated somatostatin signaling contributes to endocrine and neoplastic disease: reduced hypothalamic somatostatin tone or impaired receptor function is associated with excess growth hormone secretion in acromegaly, while high SSTR expression on neuroendocrine tumors underlies their sensitivity to somatostatin analogs that exploit the same inhibitory pathways to control hormone hypersecretion and tumor growth, and overproduction of somatostatin in somatostatinomas leads to diabetes, steatorrhea, and gallstones through marked suppression of insulin and gastrointestinal secretions.

References
[1] Patel YC. Front Neuroendocrinol. 1999 Jul;20(3):157-98. [2] Low MJ. Best Pract Res Clin Endocrinol Metab. 2004 Dec;18(4):607-22.

PVDFメンブレン孔径参考表

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

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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%