Anti dclk1 antibody

Manufactured by Abcam

Sourced in United States

Anti-DCLK1 antibody is a research-use only product that binds to the DCLK1 protein. DCLK1 is a serine/threonine protein kinase that plays a role in neuronal development and function. This antibody can be used to detect and study the DCLK1 protein in various research applications.

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Anti-DCLK1 (16 citations) DCLK1 (15 citations) DCLK1 antibody (5 citations) Anti-Dclk1 primary antibody (2 citations)

5 protocols using anti dclk1 antibody

Antibody Production and Characterization

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Polyclonal anti-mouse SPRR2A was produced in rabbits by Pacific Immunology (Ramona, CA). The synthesized peptide (CLPSVWPGP) was conjugated to a carrier protein and the conjugate was used to generate the antibody. Other antibodies were purchased from various vendors: anti-human SPRR2A antibody (Abcam, ab125385), anti-MUC2 antibody (Thermo Fisher, MA5-12345), anti-Lysozyme antibody (Santa Cruz, sc-27958), anti-DCLK1 antibody (Abcam, ab202754), anti-GAPDH antibody (Thermo Fisher, PA1-988), anti-β-actin antibody (Cell Signaling, 8457S), anti-β-tubulin (Cell Signaling, 2128S). Fluorescein isothiocyanate (FITC)-conjugated UEA-1 lectin was purchased from GeneTex (GTX01512).

Hu Z., Zhang C., Sifuentes-Dominguez L., Zarek C.M., Propheter D.C., Kuang Z., Wang Y., Pendse M., Ruhn K.A., Hassell B., Behrendt C.L., Zhang B., Raj P., Harris-Tryon T.A., Reese T.A, & Hooper L.V. (2021). Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection. Science (New York, N.Y.), 374(6568), eabe6723.

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Western Blot Analysis of EMT Markers

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Whole-cell lysates were collected using RIPA buffer. Proteins were separated using 10% SDS polyacrylamide gel, and the gels were transported to PVDF membranes (Thermo Fisher Scientific, CA, USA). The PVDF membranes were incubated with 5% skim milk in TBST at room temperature for 1 h. Later on, the PVDF membranes were probed with primary antibodies: anti-DCLK1 antibody (1:1000, Abcam, CA, USA), anti-Notch1 antibody (1:3000, Abcam, CA, USA), anti-E-cadherin (1:3000, Abcam, CA, USA), anti-Vimentin (1:3000, Abcam, CA, USA), anti-slug (1:1000, Abcam, CA, USA), anti-TGF-β (1:1000, Abcam, CA, USA), anti-MMP2 (1:1000, Abcam, CA, USA), anti-MMP9 (1:1000, Abcam, CA, USA) and anti-GAPDH antibody (1:3000, Bioworld, CA, USA) overnight at 4 °C. After that, the PVDF membrane was incubated for 1 h in secondary antibody anti-rabbit IgG second antibody (Abcam; ab150077) (1:5000) at room temperature for 1 h. Finally, the immunoreactivity was detected using ECL reagent (Santa Cruz Biotechnology).

Liu Z.Q., He W.F., Wu Y.J., Zhao S.L., Wang L., Ouyang Y.Y, & Tang S.Y. (2020). LncRNA SNHG1 promotes EMT process in gastric cancer cells through regulation of the miR-15b/DCLK1/Notch1 axis. BMC Gastroenterology, 20, 156.

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Lung Goblet Cells and Tracheal Tuft Cells

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Goblet cells were quantitated in PAS stained 2.5 μm thick sections of glycolmethacrylate-embedded lungs and eosinophil recruitment was visualized with Congo red reactivity.
For evaluation of tracheal tuft cells, tracheas were incubated with an anti-DCLK1 antibody (Abcam) for 7 days post fixation and the immunoreactivity was identified with Alexa 546 conjugated anti-rabbit antibody (Invitrogen) with Hoechst 33342 as a nuclear dye. Quantitation of tuft cells in whole mounts was as previously described (13 ).

Ualiyeva S., Lemire E., Aviles E.C., Wong C., Boyd A.A., Lai J., Liu T., Matsumoto I., Barrett N.A., Boyce J.A., Haber A.L, & Bankova L.G. (2021). Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically initiate lung type 2 inflammation. Science immunology, 6(66), eabj0474.

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Caecal Crypt and Goblet Cell Analysis

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Caecal tips were collected and fixed in Carnoy’s solution. Sections were paraffin embedded and 5μm sections were mounted onto slides for staining. To analyse caecal crypts and goblet cell counts sections were stained with periodic acid Schiff’s reagent (PAS) and counterstained with Meyers haematoxylin(Sigma). To analyse mucin sulphation, sections were stained with High- Iron Diamine-Alcian Blue (HID-AB). Images were visualised using an Axioskop upright microscope using the Axiovision software.
To stain for Brdu, Muc2 and tuft cells, sections were stained with primary anti-BRDU (BU1/75, BioRad), anti-Muc2 antibody, 1:200 dilution (5501, gift from D. Thornton) or anti-Dclk1 antibody, 1:800 dilution (Abcam) at 1:800 dilution). Sections were washed in PBS and incubated in secondary antibody goat anti-rabbit Af488 (Life techologies) for 1 hour at room temperature. Nuclear structures were stained with DAPI. An Olympus BX51 upright microscope was used to visualise staining using MetaVue software.

Glover M., Colombo S.A., Thornton D.J, & Grencis R.K. (2019). Trickle infection and immunity to Trichuris muris. PLoS Pathogens, 15(11), e1007926.

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Investigating DCLK1 and FOXD3 Regulation

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Antibodies used in these studies included: anti-β-actin (total; Sigma); anti-DCLK1 antibody (Abcam); anti-FOXD3 antibody (Abcam). Anti-DCLK1-S antibody was generated in our laboratory as described previously (9 ). The anti-DCLK1-S-Ab was specific for the S-isoform and did not cross-react with the L-isoform (9 ). Sepharose beads and all other chemical reagents were purchased from Sigma. cDNA Synthesis Master Mix was purchased from GeneDEPOT. SYBR green qRT-PCR kit was purchased from Bio-Rad. Promega GoTaq green Master Mix was used for PCR amplification, using a thermal cycler from Eppendorf. FOXD3 expression plasmid and vector controls were purchased from GeneCopoeia. Smart Pool of target-specific siRNA and nontargeting (control) siRNA were purchased from Dharmacon. Transfection reagent FuGENE6 was bought from Roche, and all primers used were synthesized by Sigma.

Sarkar S., O’Connell M.R., Okugawa Y., Lee B.S., Toiyama Y., Kusunoki M., Daboval R.D., Goel A, & Singh P. (2017). FOXD3 Regulates CSC Marker, DCLK1-S, and Invasive Potential: Prognostic Implications in Colon Cancer. Molecular cancer research : MCR, 15(12), 1678-1691.

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