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Deadpan Rat mAb

Catalog No.: F2046

Experiment Essentials

Subcellular Location: Nucleus.

Usage Information

Dilution
IHC1:800

Application
IHC

Source
Rat

Reactivity
D. Melanogaster

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
47 kDa

Positive Control	Drosophila larval brain, Drosophil embryos
Negative Control

Exprimental Methods

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
Deadpan Rat mAb detects endogenous levels of total Deadpan protein.

Uniprot ID
Q26263

Clone
K20M7

Background
Deadpan (dpn) is a transcriptional repressor belonging to the Hairy/Enhancer of Split (HES) subclass of basic Helix-Loop-Helix (bHLH) proteins in Drosophila. It functions as a key regulator of neurogenesis and sex determination by promoting the self-renewal of neural stem cells (neuroblasts) and inhibiting their differentiation. Deadpan is regulated by the Notch signaling pathway and is expressed in neural cells as well as in other tissues like wing, eye, and leg imaginal discs. Structurally, Deadpan contains a bHLH domain that facilitates DNA binding and dimerization, an Orange domain involved in protein-protein interactions, and a WRPW motif essential for transcriptional repression. Deadpan's role in repressing specific gene transcription is crucial for the proper development of neural and other tissues in Drosophila.

Background

Deadpan (dpn) is a transcriptional repressor belonging to the Hairy/Enhancer of Split (HES) subclass of basic Helix-Loop-Helix (bHLH) proteins in Drosophila. It functions as a key regulator of neurogenesis and sex determination by promoting the self-renewal of neural stem cells (neuroblasts) and inhibiting their differentiation. Deadpan is regulated by the Notch signaling pathway and is expressed in neural cells as well as in other tissues like wing, eye, and leg imaginal discs. Structurally, Deadpan contains a bHLH domain that facilitates DNA binding and dimerization, an Orange domain involved in protein-protein interactions, and a WRPW motif essential for transcriptional repression. Deadpan's role in repressing specific gene transcription is crucial for the proper development of neural and other tissues in Drosophila.

References
Deadpan (dpn) 是一种转录抑制因子，属于果蝇中基本螺旋-环-螺旋 (bHLH) 蛋白的 Hairy/Enhancer of Split (HES) 亚类。它通过促进神经干细胞 (神经母细胞) 的自我更新并抑制其分化，起到神经发生和性别决定的关键调控器的作用。Deadpan 受 Notch 信号通路调控，在神经细胞以及翅膀、眼睛和腿成虫盘等其他组织中表达。从结构上讲，Deadpan 包含一个促进 DNA 结合和二聚化的 bHLH 结构域、一个参与蛋白质-蛋白质相互作用的结构域和一个对转录抑制至关重要的 WRPW 基序。Deadpan 在抑制特定基因转录方面的作用对于果蝇神经和其他组织的正常发育至关重要。

References

Deadpan (dpn) 是一种转录抑制因子，属于果蝇中基本螺旋-环-螺旋 (bHLH) 蛋白的 Hairy/Enhancer of Split (HES) 亚类。它通过促进神经干细胞 (神经母细胞) 的自我更新并抑制其分化，起到神经发生和性别决定的关键调控器的作用。Deadpan 受 Notch 信号通路调控，在神经细胞以及翅膀、眼睛和腿成虫盘等其他组织中表达。从结构上讲，Deadpan 包含一个促进 DNA 结合和二聚化的 bHLH 结构域、一个参与蛋白质-蛋白质相互作用的结构域和一个对转录抑制至关重要的 WRPW 基序。Deadpan 在抑制特定基因转录方面的作用对于果蝇神经和其他组织的正常发育至关重要。

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

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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%