Insulin Antibody [K13G15]

카탈로그 번호 F0436

인쇄

생물학적 설명

특이성

Insulin Antibody [K13G15]는 총 Insulin 단백질의 내인성 수준을 인식합니다.
배경

인슐린은 췌장의 β세포에서 분비되는 51개의 아미노산으로 구성된 펩타이드 호르몬입니다. 이는 리간드 결합을 담당하는 두 개의 세포외 α서브유닛과 신호 전달을 매개하는 두 개의 세포내 β서브유닛으로 구성된 막관통 Protein Tyrosine Kinase인 인슐린 수용체(IR)에 결합하여 포도당 항상성을 유지하는 데 중요한 역할을 합니다. 인슐린 결합 시, 수용체는 티로신 잔기에서 자가인산화되어 키나제 활성을 활성화합니다. 이는 인슐린 수용체 기질(IRS)의 인산화로 이어지며, 이는 다시 포스포이노시티드 3-키나제(PI3K)를 모집하고 활성화합니다. 활성화된 PI3K는 포스파티딜이노시톨 (3,4,5)-트리스포스페이트(PIP3)를 생성하여 AKT(protein kinase B)의 활성화를 촉진합니다. 활성화된 AKT는 여러 하위 대사 과정을 조절합니다. 이는 GLUT4 포도당 수송체의 세포막으로의 전위를 유도하여 골격근 및 지방 조직에서 포도당 흡수를 촉진하고, 전사 인자 FOXO1을 인산화하고 억제하여 간 포도당 생성을 억제하며, 글리코겐 합성 효소 키나제 3(GSK3)을 억제하여 글리코겐 저장을 강화합니다. 또한, 인슐린 신호는 PDE3B 및 ABHD15 매개 지방세포 트리글리세리드 리파아제(ATGL) 및 호르몬 민감성 리파아제(HSL) 억제를 통해 지방분해를 억제하는 동시에 mTORC1 복합체의 활성화를 통해 지방 생성 및 단백질 합성을 촉진합니다. 인슐린 저항성은 세포가 인슐린 신호에 덜 반응하게 될 때 발생하며, 종종 경로의 여러 수준에서 손상으로 인해 발생합니다. 여기에는 인슐린 수용체 수 감소, 결함 있는 IRS 인산화 또는 PI3K/AKT 신호 교란이 포함될 수 있습니다. 기여 요인으로는 만성 염증, 산화 스트레스, 그리고 IRS-1 및 AKT 활성을 방해하는 다이아실글리세롤 및 세라마이드와 같은 지질 중간체의 축적이 있습니다. 더욱이, 간 및 골격근과 같은 조직의 이소성 지질 침착은 JNK 및 PKCθ를 포함한 스트레스 관련 키나제를 활성화하여 IRS 단백질의 세린 인산화 및 후속 인슐린 신호 억제로 이어질 수 있습니다. 미토콘드리아 기능 장애 및 소포체(ER) 스트레스 또한 손상된 포도당 대사에 기여하여 지속적인 고혈당증과 점진적인 β세포 기능 장애를 유발합니다. 이는 제2형 당뇨병의 특징입니다.

사용 정보

응용 WB, IHC, IF, FCM 희석
WB IHC IF FCM
1:200 1:6400 - 1:25600 1:200 - 1:800 1:50 - 1:200
반응성 Human, Mouse, Rat
출처 Rabbit Monoclonal Antibody MW 12 kDa
보관 완충액 PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3
보관
(수령일로부터)		 -20°C (avoid freeze-thaw cycles), 2 years
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.
 
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.
 
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.
 

참조

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

적용 데이터

WB

Selleck 검증

  • F0436-wb
    Lane 1: INS-1