Ift52

Manufactured by Proteintech

IFT52 is a laboratory equipment product designed for applications that require the detection and analysis of target proteins or molecules. The core function of this product is to facilitate the efficient isolation and purification of the desired protein or molecular species from complex biological samples.

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Lab products found in correlation

Ift81, by Proteintech (3 mentions) Ift88, by Proteintech (3 mentions) Tcs spe confocal microscope, by Leica (2 mentions) Vectashield containing 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (2 mentions) Acetylated α tubulin, by Merck Group (2 mentions) Ift140, by Proteintech (2 mentions) Kif3a, by Merck Group (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Glutathione agarose beads, by Merck Group (1 mentions) Anti flag m2 resin, by Merck Group (1 mentions) Lamin b1, by Proteintech (1 mentions) Gm130, by BD (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Gapdh, by Proteintech (1 mentions) α tubulin, by Merck Group (1 mentions) Arl13b, by Proteintech (1 mentions)

4 protocols using ift52

Quantitative Immunoblotting of Cilia Proteins

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Each antibody was tested in at least three experimental replicates from independent cultures. Cell lysates were collected from 80% confluence starved cultures in RIPA buffer with proteinase inhibitors (SIGMA). Lysates were cleared by centrifugation and quantified (Pierce BCA protein assay kit) to equal loading. 40 ug of each lysate were loaded with Laemmli buffer and run by 10% acrylamide SDS-PAGE gels and transfer to PVDF membranes. Membranes were saturated for 1 h and antibody-incubated overnight in 5% milk in TBST. HRP secondary antibody and ECL-film exposure was used to detect bands. Antibodies used were: IFT81 (Proteintech 1:1000); CMG or IFT74 (Abcam 1:500); IFT52 (Proteintech 1:1000); IFT88 (Proteintech 1:500); Aceto-Tubulin (SIGMA T6793 1:2000); KIF3A (Sigma 1:2000) Gli3 (R&D 1:200); GAPDH (Cell Signaling 1:1000). HRP secondary antibodies were Cell Signaling (1:1000, anti-mouse and–rabbit) and Santa Cruz (1:3000, anti-goat). FIJI was used to quantify bands following Gel Analysis recommendations from ImageJ and Gassmann et al. (http://rsb.info.nih.gov/ij/docs/menus/analyze.html#gels)60 (link) and Mann-Whitney test was performed for statistic analysis using Prism software.

Duran I., Taylor S.P., Zhang W., Martin J., Forlenza K.N., Spiro R.P., Nickerson D.A., Bamshad M., Cohn D.H, & Krakow D. (2016). Destabilization of the IFT-B cilia core complex due to mutations in IFT81 causes a Spectrum of Short-Rib Polydactyly Syndrome. Scientific Reports, 6, 34232.

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Immunofluorescence Localization of Ciliary Proteins

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Cells were washed with PBS, fixed with 4% paraformaldehyde and 0.2% triton X-100 for 10 minutes at room temperature, washed with PBS, and blocked with 1% BSA in PBS for 1 hour, and then incubated with antibodies against SMO (generous gift from Dr. K Anderson, and purchased from Abcam), IFT52, IFT81, IFT88, IFT140, BBS2, BBS5 (Proteintech) and acetylated α-tubulin (Sigma) overnight at 4 °C. To address localization of the SMO within cilia, cells were first treated with 500 nM SAG (Enzo Life Sciences) in DMSO. Following 3 washes in PBS, cells were incubated with anti-rabbit AF488 and anti-mouse AF594 (InVitrogen Technologies) for 30 minutes at room temperature. Cells were washed 3X in PBS, and mounted with Vectashield containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Immuno-labeled cells were viewed and imaged using a Leica TCS SPE confocal microscope configured on a DM550 Q upright microscope. Each ciliary antibody was examined in a minimum of three NHK and three ADPKD cell lines.

Silva L.M., Jacobs D.T., Allard B.A., Fields T.A., Sharma M., Wallace D.P, & Tran P.V. (2018). Inhibition of Hedgehog signaling suppresses proliferation and microcyst formation of human Autosomal Dominant Polycystic Kidney Disease cells. Scientific Reports, 8, 4985.

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Antibody Generation for Wdr78, Dnah2, and Plk4 Proteins

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Rabbit antibody against mouse Wdr78 or Dnah2 were generated against peptide YLETYRGHKGPVYK or YGKLRDTIEQEIR, respectively, and affinity-purified (Abgent) for immunoblotting and immunofluorescence staining. Rabbit antibody against mouse Wdr78 was raised using bacterially expressed His-Wdr78 (1–420 amino acids) and then affinity-purified using GST-Wdr78 (1–420 amino acids) for IP. Rabbit anti-Plk4 antibody was prepared as described (Zhao et al., 2013 (link)). Commercial mouse monoclonal antibodies used were acetylated tubulin (Sigma, T6793), GST (Wolwo, Mab-GS01), His (Sigma, H1029), Tctex1 (Millipore, MAB1076), DIC74.1 (Millipore, MAB1618), Dnai2 (Abnova, H00064446-M01), and GM130 (BD, 610823). Commercial rabbit antibodies used were Flag (Sigma, F7425), Ift80 (GeneTex, GTX109393), Ift52 (Proteintech, 17534-1-AP), Rsph3 (Proteintech, 17603-1-AP), GFP (Life Technologies, A6455), Wdr63 (GeneTex, GTX45697), Lamin B1 (Proteintech,12987-1-AP), and Gapdh (Proteintech, 10494-1-AP). Secondary antibodies conjugated with peroxidase or Alexa Fluor-488, -546, or -647 were purchased from Life Technologies. Conformation-specific mouse anti-rabbit IgG mAb conjugated with peroxidase was purchased from Cell Signaling Technology (L27A9). Anti-Flag M2 resin and glutathione agarose beads were from Sigma. Recombinant protein G (rProtein G) agarose beads were from Life Technologies.

Zhang Y., Chen Y., Zheng J., Wang J., Duan S., Zhang W., Yan X, & Zhu X. (2018). Vertebrate Dynein-f depends on Wdr78 for axonemal localization and is essential for ciliary beat. Journal of Molecular Cell Biology, 11(5), 383-394.

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Immunofluorescent Labeling of Ciliary Proteins

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Cells were washed with PBS, then fixed with 4% paraformaldehyde/0.2% triton X-100 in PBS for 10 minutes at room temperature. Cells were washed again with PBS, and blocked with 1% BSA in PBS for 1 hour. Cells were then incubated with antibodies against SMO (Abcam), IFT52, IFT81, IFT88, IFT140, BBS2, BBS5 (Proteintech), ARL13B and INNP5E (Proteintech) and α-tubulin and acetylated α-tubulin (Sigma), and polyglutamylated tubulin (Adipogen) overnight at 4° C. Following 3 washes in PBS, cells were incubated with anti-rabbit AF488 and anti-mouse AF594 (InVitrogen Technologies) for 30 minutes at room temperature. Cells were washed 3X in PBS, and mounted with Vectashield containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Immuno-labeled cells were viewed and imaged using a Leica TCS SPE confocal microscope configured on a DM550 Q upright microscope.

Wang W., Allard B.A., Pottorf T.S., Wang H.H., Vivian J.L, & Tran P.V. (2020). Genetic interaction of mammalian IFT-A paralogs regulates cilia disassembly, ciliary entry of membrane protein, Hedgehog signaling and embryogenesis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(5), 6369-6381.

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