CENPF Antibody (Rabbit mAb) [G20B19]

Catalog No.: F3738

Application: Reactivity: Human

当該製品は品切れ状态で、メールアドレスをご教示いただければ、お客様に返信いたします。

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

使用情報

Dilution
IHC1:1000 IF1:100

Application
IHC, IF

Source
Rabbit Monoclonal Antibody

Reactivity
Human

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
357 kDa

ポジティブコントロール	Human breast cancer tissue; Human tonsil tissue; A549 cells; HeLa cells
ネガティブコントロール

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
CENPF Antibody (Rabbit mAb) [G20B19] detects endogenous levels of total CENPF protein.

Uniprot ID
P49454

Clone
G20B19

Synonym(s)
Centromere protein F, CENP-F, AH antigen, Kinetochore protein CENPF, Mitosin, CENPF

Background
Centromere protein F (CENPF, mitosin) is a large, cell‑cycle‑regulated nuclear matrix and kinetochore protein that functions as a dynamic scaffold coordinating chromosome alignment, kinetochore–microtubule attachment, spindle checkpoint signaling, and cell‑cycle progression at the G2/M transition. The polypeptide is predominantly coiled‑coil and forms dimers that accumulate on the nuclear matrix in late G2, then relocalize to outer kinetochores from prophase through early anaphase, before shifting to the spindle midzone and intercellular bridge in late anaphase and telophase, where it is subsequently degraded, reflecting tight temporal control linked to mitotic events. CENPF provides a docking platform at kinetochores for multiple microtubule‑associated and checkpoint proteins, including the motor CENP‑E and the dynein/LIS1/NDE1/NDEL1 complex, and contributes to recruitment or stabilization of Bub1 and related spindle checkpoint components, thereby promoting robust kinetochore–microtubule capture, chromosome congression, tension sensing, and proper activation and silencing of the spindle assembly checkpoint. Loss or silencing of CENPF weakens centromeric cohesion, delays or prevents stable bioriented attachments and metaphase plate formation, prolongs mitosis, and triggers persistent spindle checkpoint activation, leading to chromosome missegregation and aneuploidy, which identifies CENPF as a core factor for accurate chromosome segregation. CENPF interacts with DNA‑PK and contributes to interphase chromatin organization and DNA synthesis control, and its C‑terminal region modulates pocket protein (RB family) activity to influence cell‑cycle progression and lineage decisions, including roles in embryonic cardiomyocyte cycling, skeletal myogenesis, and cardiac lineage specification from embryonic stem cells. Additional interactions with syntaxin‑4 and SNAP25 connect CENPF to recycling vesicles and the microtubule network, implicating it in membrane trafficking and plasma‑membrane recycling during cell division. Expression of CENPF is low in quiescent cells and peaks in proliferating and mitotic cells, and high CENPF levels are observed in a broad range of tumors where they correlate with poor prognosis, high grade, and features of chromosomal instability, while functional studies in hepatocellular carcinoma and prostate cancer indicate that CENPF promotes G2/M transition, drives proliferation, and can modulate metabolic pathways such as pyruvate kinase M2 phosphorylation signaling.

Background

Centromere protein F (CENPF, mitosin) is a large, cell‑cycle‑regulated nuclear matrix and kinetochore protein that functions as a dynamic scaffold coordinating chromosome alignment, kinetochore–microtubule attachment, spindle checkpoint signaling, and cell‑cycle progression at the G2/M transition. The polypeptide is predominantly coiled‑coil and forms dimers that accumulate on the nuclear matrix in late G2, then relocalize to outer kinetochores from prophase through early anaphase, before shifting to the spindle midzone and intercellular bridge in late anaphase and telophase, where it is subsequently degraded, reflecting tight temporal control linked to mitotic events. CENPF provides a docking platform at kinetochores for multiple microtubule‑associated and checkpoint proteins, including the motor CENP‑E and the dynein/LIS1/NDE1/NDEL1 complex, and contributes to recruitment or stabilization of Bub1 and related spindle checkpoint components, thereby promoting robust kinetochore–microtubule capture, chromosome congression, tension sensing, and proper activation and silencing of the spindle assembly checkpoint. Loss or silencing of CENPF weakens centromeric cohesion, delays or prevents stable bioriented attachments and metaphase plate formation, prolongs mitosis, and triggers persistent spindle checkpoint activation, leading to chromosome missegregation and aneuploidy, which identifies CENPF as a core factor for accurate chromosome segregation. CENPF interacts with DNA‑PK and contributes to interphase chromatin organization and DNA synthesis control, and its C‑terminal region modulates pocket protein (RB family) activity to influence cell‑cycle progression and lineage decisions, including roles in embryonic cardiomyocyte cycling, skeletal myogenesis, and cardiac lineage specification from embryonic stem cells. Additional interactions with syntaxin‑4 and SNAP25 connect CENPF to recycling vesicles and the microtubule network, implicating it in membrane trafficking and plasma‑membrane recycling during cell division. Expression of CENPF is low in quiescent cells and peaks in proliferating and mitotic cells, and high CENPF levels are observed in a broad range of tumors where they correlate with poor prognosis, high grade, and features of chromosomal instability, while functional studies in hepatocellular carcinoma and prostate cancer indicate that CENPF promotes G2/M transition, drives proliferation, and can modulate metabolic pathways such as pyruvate kinase M2 phosphorylation signaling.

References
[1] Shahid M, et al. Cell Cycle. 2018;17(24):2802-2818. [2] Dai Y, et al. Biochem Biophys Res Commun. 2013 Jul 12;436(4):711-8.

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

技術サポート

ストックの作り方、阻害剤の保管方法、細胞実験や動物実験の際に注意すべき点など、製品を取扱う時に問い合わせが多かった質問に対しては取扱説明書でお答えしています。

Handling Instructions

他に質問がある場合は、お気軽にお問い合わせください。

* 必須

* 大学・企業名 大学・企業名を記入してください

* 名前 名前を記入してください

* 電子メール 電子メール・アドレスを記入してください 有効なメールアドレスを入力してください

電話番号

* 内容 お問い合わせ内容をご入力ください

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%