Figure 1: Structure of the Doublecortin target protein.

Doublecortin Target Introduction

Protein Function

• Doublecortin is a member of the doublecortin family, containing two doublecortin domains that can bind to microtubules.
• During brain cortex development, doublecortin can regulate the organization and stability of microtubules and bundle them together to guide neuronal migration.
• Mutations in doublecortin lead to abnormal neuronal migration during development and disrupt the layering of the cerebral cortex, which is associated with diseases such as female epilepsy, cognitive impairment, subcortical band heterotopia, and male lissencephaly (smooth brain syndrome).

Protein Expression

• Doublecortin is highly expressed in neuronal cells of the fetal brain (cortical plate, intermediate zone, and most cells in the ventricular zone) but not expressed in other fetus tissues.
• Doublecortin is highly expressed in the frontal lobe of the adult brain but expressed very low in other brain regions. It is not detected in the heart, placenta, lungs, liver, skeletal muscle, kidneys, and pancreas.

Protein Characteristics

• Doublecortin expression is tissue-specific, so please choose appropriate experimental samples.

Protein Localization

• Doublecortin is mainly localized in the cytoplasm but can also be found in projections of neuronal cells (axons and dendrites).

Figure 2: Doublecortin ICC experiment result image, recombinant Anti-Doublecortin antibody [EPR19997] (ab207175)

Sample name: SH-SY5Y (human neuroblastoma cell line derived from bone marrow)
Green: Doublecortin, Red: Tubulin, Blue: DAPI
Experimental result: Confocal image shows cytoplasmic staining of SH-SY5Y cell line.

Isoforms & Post-translational modifications

• Human (O43602): Isoform 1-2: 40-41 kDa (predicted)
• Mouse (O88809): 41 kDa (predicted)
• Rat (Q9ESI7): 41 kDa (predicted)
• Doublecortin can be phosphorylated by MARK1, MARK2, and PKA, thereby regulating its ability to bind to microtubules.
• Phosphorylation of the serine residues Ser-265 and Ser-297 of Doublecortin appears to occur only in the neonatal brain, sharply decreasing by day 21 after birth.
• Doublecortin can be ubiquitinated by MDM2, leading to its degradation by the proteasome.

IHC experiment tips

Precautions

• Doublecortin is specifically expressed in cells in fetal brain and other tissues. Please select appropriate experimental samples, as some samples may show weak or no expression.
• After fixing the tissue with aldehydes, please refer to the antibody product instructions for appropriate antigen retrieval.
• Positive and negative controls should be set for each round of experiments. The positive control ensures the effectiveness of the primary/secondary antibodies, and the negative control is the secondary antibody control without the primary antibody.

Positive control

• Adult rat/mouse hippocampal tissue.

Negative control (weak or no expression)

• Mouse heart tissue.

Example of results

Figure 3: Recombinant Anti-Doublecortin antibody [EPR19997] (ab207175)

Sample name: Frozen sections of adult rat hippocampal tissue
Experimental results: Cytoplasmic staining of the dentate gyrus in the mouse hippocampus was observed (consistent with literature PMID: 23690918, 16814555)
Green: Doublecortin, Blue: DAPI

Figure 4: Recombinant Anti-Doublecortin antibody [EPR19997] (ab207175)

Sample name: Paraffin-embedded adult mouse hippocampal tissue
Antigen retrieval method: Heat-induced antigen retrieval using pH 9 Tris/EDTA buffer
Experimental results: Cytoplasmic staining of the dentate gyrus in the mouse hippocampus was observed (consistent with literature PMID: 23690918, 16814555)

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample fixation:

1. The fixation time of the sample depends on the size of the tissue block and the type of tissue, but for most samples, such as fixation with 4% PFA, it is more appropriate to fix at room temperature for 18-24 hours.
2. Insufficient fixation will result in higher signal at the edges of the sample and weaker signal at the center, or even no signal.
3. Excessive fixation will block antigenic epitopes. Although antigen retrieval will expose some of the epitopes, if the tissue fixation time is very long (more than a week), there may still be no signal after antigen retrieval.

Blocking:

1. If using fluorescently conjugated secondary antibodies for the experiment, we recommend using a blocking solution containing 1% BSA and a final concentration of 0.3 M glycine to quench the autofluorescence caused by aldehyde groups.
2. If using HRP conjugates for detection, please use 3% hydrogen peroxide to treat the sections for 10 minutes to block endogenous peroxidase.
3. Before incubating with the primary antibody, it is necessary to block with serum and avoid using blocking solutions from the same species as the host. The source of the serum can be selected based on the host of the secondary antibody. For example, if the secondary antibody is Goat Anti-Rabbit IgG H&L (HRP polymer) or Goat Anti-Mouse IgG H&L (HRP polymer), goat serum can be used as the blocking solution.

Antigen retrieval:

1. When performing immunohistochemistry on paraffin sections, we recommend using a pressure cooker for heat-induced antigen retrieval. You can try fixing the sections at 110°C for 15 minutes.
2. When performing immunohistochemistry on frozen sections, if the samples have been fixed with aldehydes for 18-24 hours in the preliminary stage, you can try using a microwave for short-term retrieval or using enzymes for antigen retrieval. However, please optimize the enzyme concentration and retrieval time to avoid damaging the tissue morphology of the sections.

WB experiment tips

Precautions

• Doublecortin is highly expressed during embryonic development and downregulated in adult tissues. Some samples may show weak or no expression. Please choose appropriate experimental samples and use positive controls to confirm the experimental system is working properly.
• Due to the phosphorylation modification of doublecortin, multiple bands may be detected in the WB experiment.
  Do not strip the membrane or at least retain the part below 70 kDa to prevent signal loss.

Positive controls

• SH-SY5Y, IMR-32.
• Human fetal brain lysate.

Negative controls (weak or no expression)

• Human fetal heart lysate.

Example of results.

Figure 5: Recombinant Anti-Doublecortin antibody [EPR19997] (ab207175)

Lane 1: Human fetal brain lysate
Lane 2: Human brain lysate

Predicted band size: 40 kDa
Experimental results: The higher molecular weight band represents the phosphorylated form, while the lower molecular weight band represents the non-phosphorylated form (PMID: 17178868)

*Doublecortin is highly expressed during embryonic development and downregulated in adult tissues.

Figure 6: Recombinant Anti-Doublecortin antibody [EPR19997] (ab207175)

Lane 1: Human fetal brain lysate
Lane 2: Human fetal heart lysate
Lane 3: Mouse brain lysate
Lane 4: Mouse heart lysate
Lane 5: Rat brain lysate
Lane 6: Rat heart lysate

Predicted band size: 40 kDa
Experimental results: The observed expression pattern is consistent with the description in the literature (PMID: 10550327)

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex protease inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.

Transfer:

1. We recommend using Coomassie Brilliant Blue staining after transfer to confirm the success of the transfer.
2. We recommend not cutting the membrane.

References

1. Mercedes F Paredes, David James, Sara Gil-Perotin, Hosung Kim, Jennifer A Cotter, Carissa Ng, Kadellyn Sandoval, David H Rowitch, Duan Xu, Patrick S McQuillen, Jose-Manuel Garcia-Verdugo, Eric J Huang, Arturo Alvarez-Buylla. Extensive migration of young neurons into the infant human frontal lobe. Science. 2016 Oct 7;354(6308):aaf7073. doi:10.1126/science.aaf7073.
2. Xavier H Jaglin, Jamel Chelly. Tubulin-related cortical dysgeneses: microtubule dysfunction underlying neuronal migration defects. Trends Genet. 2009 Dec;25(12):555-66. doi: 10.1016/j.tig.2009.10.003.
3. Gerd Kempermann, Fred H Gage, Ludwig Aigner, Hongjun Song, Maurice A Curtis, Sandrine Thuret, H Georg Kuhn, Sebastian Jessberger, Paul W Frankland, Heather A Cameron, Elizabeth Gould, Rene Hen, D Nora Abrous, Nicolas Toni, Alejandro F Schinder, Xinyu Zhao, Paul J Lucassen, Jonas Frisén. Human Adult Neurogenesis: Evidence and Remaining Questions. Cell Stem Cell. 2018 Jul 5;23(1):25-30. doi: 10.1016/j.stem.2018.04.004. Epub 2018 Apr 19.
4. Myung Hee Kim, Tomasz Cierpicki, Urszula Derewenda, Daniel Krowarsch, Yuanyi Feng, Yancho Devedjiev, Zbigniew Dauter, Christopher A Walsh, Jacek Otlewski, John H Bushweller, Zygmunt S Derewenda. The DCX-domain tandems of doublecortin and doublecortin-like kinase. Nat Struct Biol. 2003 May;10(5):324-33. doi: 10.1038/nsb918.
5. Y Feng, C A Walsh. Protein-protein interactions, cytoskeletal regulation and neuronal migration. Nat Rev Neurosci. 2001 Jun;2(6):408-16. doi: 10.1038/35077559.
6. Dominique Burger, Martine Stihle, Ashwani Sharma, Paola Di Lello, Jörg Benz, Brigitte D'Arcy, Maja Debulpaep, David Fry, Walter Huber, Thomas Kremer, Toon Laeremans, Hugues Matile, Alfred Ross, Arne C Rufer, Guillaume Schoch, Michel O Steinmetz, Jan Steyaert, Markus G Rudolph, Ralf Thoma, Armin Ruf. Crystal Structures of the Human Doublecortin C- and N-terminal Domains in Complex with Specific Antibodies. J Biol Chem. 2016 Jul 29;291(31):16292-306. doi: 10.1074/jbc.M116.726547. Epub 2016 May 10.