SCG10/Stathmin-2 Antibody [D11B18]

카탈로그 번호 F4944

인쇄

생물학적 설명

특이성

SCG10/Stathmin-2 Antibody [D11B18]는 총 SCG10/Stathmin-2 단백질의 내인성 수준을 검출합니다.
배경

SCG10 (Stathmin-2, STMN2)은 뉴런에 풍부한 미세소관 불안정화 단백질로, 스타스민 계열에 속하며 축삭 성장 및 재생에 필수적입니다. 이 단백질은 JNK1, MAPK, PKA와 같은 키나아제의 표적이 되는 4개의 주요 인산화 부위(Ser22, Ser25, Ser38, Ser63)를 포함하는 N-말단 조절 도메인을 가지고 있습니다. C-말단 영역은 두 개의 튜불린 결합 포켓을 갖는 α-나선형 스타스민 유사 도메인을 특징으로 하며, 이는 SCG10이 α/β-튜불린 이종이합체를 1:2의 비율로 격리할 수 있도록 합니다. 이러한 격리는 종방향 원섬유 조립을 방지하여 미세소관 중합을 억제합니다. 탈인산화된 상태에서 SCG10은 자유 튜불린에 높은 친화도(Kd ~0.3–1 μM)로 결합하여 GTPase-가능 튜불린을 포획함으로써 동역학을 탈중합화 방향으로 이동시켜 미세소관 파괴를 촉진합니다. 그러나 SCG10의 인산화는 음전하를 도입하고 튜불린 결합을 방해하는 형태 변화를 유도하여 미세소관 성장을 위한 튜불린 이종이합체를 방출합니다. 이 메커니즘은 성장 중 축삭 미세소관을 안정화시킵니다. JNK1 매개 Ser22/Thr22 인산화는 피질 뉴런 이동 동안 특히 중요하며, 다극성에서 양극성 형태 전환을 조절하고 방사형 이동 속도를 조절합니다. SCG10 발현은 발달 중인 CNS 뉴런에서 가장 높으며, 여기서 칼미린 결합을 통해 Ca²⁺ 의존적 미세소관 리모델링을 매개하여 성장 원추 역학을 촉진하고 손상 후 축삭 재생에 기여합니다. ALS/FTD에서 TDP-43의 손실은 STMN2 전사체에 암호화된 엑손 포함을 유발하여 기능성 SCG10 단백질의 고갈을 초래합니다. 이는 축삭 퇴행, 신경근 접합부 수축 및 운동 결함을 유발하며, 완전 길이 STMN2 발현을 회복함으로써 이러한 표현형을 역전시킬 수 있습니다.

사용 정보

응용 IP, IHC 희석
반응성 Human, Mouse, Rat
출처 Mouse Monoclonal Antibody MW ˜20 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
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/37236359/
  • https://pubmed.ncbi.nlm.nih.gov/39253877/

적용 데이터