Mab414

Manufactured by Abcam

Sourced in United Kingdom

Mab414 is a monoclonal antibody targeted against the nup62 protein, a component of the nuclear pore complex. It can be used for the detection and localization of nup62 in various applications such as Western blotting, immunohistochemistry, and immunocytochemistry.

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Lab products found in correlation

Alexa fluor 488, by Thermo Fisher Scientific (3 mentions) Alexa fluor rhodamine red x, by Jackson ImmunoResearch (2 mentions) Alexa fluor 647, by Jackson ImmunoResearch (2 mentions) Tcs sp5, by Leica camera (2 mentions) β actin monoclonal antibody, by Merck Group (2 mentions) His tag monoclonal antibody, by Takara Bio (2 mentions) Donkey anti rabbit and sheep anti mouse, by GE Healthcare (2 mentions) Mouse anti nxf1 antibody, by Merck Group (2 mentions) Mouse anti m2 antibody, by Thermo Fisher Scientific (2 mentions) Rabbit anti ha antibody, by GeneTex (2 mentions) Rabbit anti na antibody, by GeneTex (2 mentions) Goat anti influenza a virions antibody, by Meridian Bioscience (2 mentions) Anti flag m2 monoclonal antibody, by Merck Group (2 mentions) Mouse igg, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions) Ecl reagent, by Thermo Fisher Scientific (1 mentions) Nf κb p65, by Santa Cruz Biotechnology (1 mentions) Nup358, by Abcam (1 mentions) Rangap1, by Santa Cruz Biotechnology (1 mentions) C jun, by Cell Signaling Technology (1 mentions)

21 protocols using mab414

Western Blot Analysis of Protein Extracts

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Protein extracts were treated as previously described [5 (link)]. Briefly, between 25 and 40μg of proteins were separated by SDS-PAGE (8%, 10% or 12% acrylamide), and transferred to PVDF membranes. Membranes were blocked in Tris Buffered Saline and tween 0.1% (TBS-T) containing 5% BSA for 1 hr and incubated either 2 hrs at room temperature or over-night at 4°C with primary antibody. After washing with TBS-T (2 times for 5min), membranes were incubated 1 hr with secondary anti-HRP-conjugated antibody (GE Healthcare, Mississauga, ON, Canada). After washing with TBS-T (3 times for 5min), they were developed by chemoluminescence immunodetection with ECL reagents (Thermo Fisher Scientific, Rockford, IL) and autoradiography.
List of antibodies used: GP63 monoclonal antibody clone #253 (Button et al. 1991); Histone H2B (Genscript corporation, A01174); KDEL (ab12223), SHP-1 (ab3254), NPC Mab414 (ab24609), Nup62 (ab96134), Nup358 (RanBP2, ab64276), KpnB1 (ab2811), KpnA3 (ab105348) from Abcam; PGK1 (ProteinTech, 17811-1-AP); Actin (Sigma, A5316); TCPTP (MediMabs, MM-0018-P); NF-κB p65 (SC-8008), Nup93 (SC-374400), Nup214 (SC-26055), RanGAP1 (SC-1862), Kif1B (SC-28540) from Santa Cruz; C-Jun (Cell Signaling, 60A8).

Isnard A., Christian J.G., Kodiha M., Stochaj U., McMaster W.R, & Olivier M. (2015). Impact of Leishmania Infection on Host Macrophage Nuclear Physiology and Nucleopore Complex Integrity. PLoS Pathogens, 11(3), e1004776.

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Staining Nuclear Pore Complex in Cells

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For nuclear pore complex staining in HEK293T, cells were fixed with 4% Paraformaldehyde (Electron Microscopy Sciences), then a 5-minute incubation in 0.5% Triton-X-PBS for cell permeabilization. For fixation of hematopoietic cells and NUP98-KDM5A PDX cells, a cytocentrifuge was used to adhere cells to a glass slide by spinning at 400 rpm for 4 minutes. The cells were then rapidly rinsed in 1X PBS-5mM EGTA, followed by incubation at −20 °C in 95% Methanol-5 mM EGTA for 30 minutes. The primary antibodies used were Mouse anti-NUP107 (Abcam, Mab414, RRID:AB_448181, 1:300) and Rat anti-NUP98 (GeneTex, 2H10, RRID: AB_2894964, 1:200). The secondary antibodies used were raised in Donkey and conjugated to Alexa Fluor Rhodamine Red^™-X or Alexa Fluor 647 (Jackson ImmunoResearch; RRID:AB_2340614) at 1:300 diluted in 5% Normal Donkey Serum. Cells were counter stained with DAPI (4’,6-Diamidino-2-Phenylindole, Dihydrochloride, Invitrogen) diluted in PBS (300 nM) for 2 minutes, and then mounted onto glass slides with antifade solution (90% glycereol, 0.5% N-propyl gallate). See Suppl. Methods for additional details.

Chandra B., Michmerhuizen N.L., Shirnekhi H.K., Tripathi S., Pioso B.J., Baggett D.W., Mitrea D.M., Iacobucci I., White M.R., Chen J., Park C.G., Wu H., Pounds S., Medyukhina A., Khairy K., Gao Q., Qu C., Abdelhamed S., Gorman S.D., Bawa S., Maslanka C., Kinger S., Dogra P., Ferrolino M.C., Di Giacomo D., Mecucci C., Klco J.M., Mullighan C.G, & Kriwacki R.W. (2021). Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation. Cancer Discovery, 12(4), 1152-1169.

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Immunofluorescence Staining of Cultured Cells

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Fixation of cultured cells grown on coverslips was with 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min followed by permeabilization with 0.5% Triton X-100 for 5 min for primary antibody staining. Alternatively, cells were ﬁxed in cold methanol (−20°C) for 10 min. The following antibodies were used: rabbit monoclonal anti-SUN1 (Abcam), rabbit monoclonal anti-SUN2 (Abcam), mouse monoclonal anti-hnRNP F/H (ImmuQuest), mouse monoclonal anti-hnRNP K/J (ImmuQuest), rabbit polyclonal anti-Lamin B1 (Abcam), mouse monoclonal anti-NXF1 (Abcam), mouse monoclonal anti-RUVBL1 (Abcam), mouse monoclonal anti-Nuclear Pore Complex Proteins (mAb414) (Abcam), mouse monoclonal anti-Nup153 [QE5] (Abcam), rabbit polyclonal anti-GANP (Bethyl Laboratories) and mouse monoclonal anti-GAPDH−POD (Sigma). The secondary antibodies used were conjugated with Alexa Fluor 488 and Alexa Fluor 568 (Molecular Probes, Sigma). The samples were counterstained with DAPI (Sigma) for DNA and mounted in gelvatol. Samples were analyzed using confocal laser scanning microscopy (TCS-SP5, Leica) or Leica Structured Illumination (integration of the OptiGrid^® module, Leica research microscopes and Leica MM AF software).

Li P, & Noegel A.A. (2015). Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export. Nucleic Acids Research, 43(20), 9874-9888.

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Immunofluorescence Staining of Cellular Structures

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For immunofluorescence, cells were plated on 18 mm/22 mm glass coverslips(Himedia) in 6 well culture dishes (nunc) one day before transfection. 48 hrs post transfection cells were washed with 1x PBS for 2 times 1 min each at room temperature. Followed by PBS washing cells were fixed with 4%Paraformaldehyde for 10 min at RT and permeabilized with 0.5% Triton X-100 for 5 min at Rt. Before and after cell fixation and permeabilization cells were washed with 1x PBS for 5 times 3 min each. Thereafter, cells were incubated with primary antibody (abcam anti-Lamin B1 antibody [ab16048] 1:500 dilution, abcam anti-nuclear pore complex proteins antibody [Mab 414] 1:1000 dilution for 2 hrs at RT. Followed by incubation with primary antibody cells were washed with PBS / 0.05% Tween 20 buffer and PBS 3 times at room temperature. Then cells were incubated with secondary antibody goat Anti-mouse IgG (H + L) secondary antibody, Alexa Fluor 594 conjugate Invitrogen (A11005) antibody for 1 hr at RT. Finally, cells were again washed with washing buffer and PBS 3 times each for 3 min at RT. Coverslips were mounted with Vectashield containing DAPI for staining DNA on glass slides (Himedia). At final stage, coverslips were sealed with transparent nail paint.

Dutta S., Das J.K., Maganti L., Bhattacharyya M., Bhattacharyya D., Mukherjee S, & Sengupta K. (2018). Skeletal Muscle Dystrophy mutant of lamin A alters the structure and dynamics of the Ig fold domain. Scientific Reports, 8, 13793.

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Germline Immunofluorescence Staining Protocol

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Germlines from young adult worms were dissected on glass slides and fixed with 2% paraformaldehyde for 10 min at room temperature. Fixed germlines were freeze cracked on dry ice and immersed in 100% methanol for 5 min. The germlines were incubated with a 1:1 methanol:acetone solution for 5 min then transferred to 100% acetone for another 5 min. Fixed germlines were washed in 1× PBS/0.1% Tween20 (PBST) four times for 10 min each. The samples were incubated in 1 drop of Image-iT (Thermo Fisher Scientific) for 20 min and blocked in PBST/1% BSA for 1 h. phospho-MPK-1 was probed with monoclonal α-p-ERK antibody (Cell Signalling #4370) and monoclonal α-Nuclear Pore Complex antibody (Mab414, Abcam) in PBST/1% BSA overnight at room temperature. Germlines were washed in PBST three times for 10 min each and probed with goat α-rabbit-conjugated Alexa Fluor 488 (Thermo Fisher Scientific) and donkey α-mouse Alexa Fluor 568 at room temperature for 1 h. Stained germlines were then washed with PBST for 10 min and nuclear DNA was stained with DAPI. Samples were coated with Prolong Gold (Thermo Fisher Scientific) and imaged as described above.

Tran A.T., Chapman E.M., Flamand M.N., Yu B., Krempel S.J., Duchaine T.F., Eroglu M, & Derry W.B. (2019). MiR-35 buffers apoptosis thresholds in the C. elegans germline by antagonizing both MAPK and core apoptosis pathways. Cell Death and Differentiation, 26(12), 2637-2651.

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Antibody Dilutions for Influenza A Virus Detection

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Influenza A virus NS1 antibody, a gift from J.A. Richt (National Animal Disease Center, Iowa)^{28 (link)} was used at a 1:10000 dilution. β-actin monoclonal antibody (Sigma-Aldrich, clone AC-74, A5528) was used at a 1:5000 dilution. His tag monoclonal antibody (TAKARA & Clontech, 631210) was used at a 1:5000 dilution. ANTI-FLAG® M2 monoclonal antibody (Sigma-Aldrich, F1804) was used at a 1:1000 dilution. Horseradish peroxidase (Hrp)-conjugated secondary antibodies included donkey anti-rabbit and sheep anti-mouse (GE Healthcare, NA934V and NA931V, respectively). Polyclonal rabbit anti-Nup98 antibody^{29 (link)} was used at a 1:1000 dilution. Mouse anti-nuclear pore complex proteins antibody MAB414 (Abcam, ab24609) was used at a 1:1000 dilution. Mouse anti-NXF1 antibody (Sigma-Aldrich, T1076–200UL) was used at a 1:2000 dilution. Mouse anti-M2 antibody (Thermo Fisher Scientific, MA1–082) was used at a 1:1000 dilution. Rabbit anti-HA antibody (Genetex, GTX127357) was used at a 1:1000 dilution. Rabbit anti-NA antibody (Genetex, GTX125974) was used at a 1:1000 dilution. Goat anti-influenza A virions antibody (Meridian Life Science, B65141G) was used at a 1:1000 dilution. Mouse IgG (Thermo Fisher Scientific, 31903) was diluted to 0.4 mg/ml.

Zhang K., Xie Y., Muñoz-Moreno R., Wang J., Zhang L., Esparza M., García-Sastre A., Fontoura B.M, & Ren Y. (2019). Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export. Nature microbiology, 4(10), 1671-1679.

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Immunofluorescence Imaging of Cellular Structures

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LnCAP or RWPE-1 cells were seeded in glass chamber slides and siRNA was performed as described above. After five days, cells were fixated with 1.6% paraformaldehyde, blocking was performed using 5% goat serum (Abcam, Cambridge, UK) and 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in TBS. Cells were incubated with primary antibodies targeting β-tubulin (9F3; Cell Signalling Technology, Danvers, MA, USA) or nuclear pore complex proteins (Mab414, Abcam) o/n at 4 °C. Incubation with the secondary antibodies (Alexa Fluor^® 488 Goat anti-rabbit, Abcam; Alexa Fluor^® 546 Goat anti-mouse, Invitrogen) was performed for 1 h at RT. Before mounting the samples with Fluoroshield^TM (Sigma-Aldrich), DAPI staining was performed for 3 min at RT. Images were taken using a NIKON C2 Eclipse Ti microscope (Nikon, Tokyo, Japan).

Ertl I.E., Brettner R., Kronabitter H., Mohr T., Derdak S., Jeitler M., Bilban M., Garstka N, & Shariat S.F. (2022). The SMARCD Family of SWI/SNF Accessory Proteins Is Involved in the Transcriptional Regulation of Androgen Receptor-Driven Genes and Plays a Role in Various Essential Processes of Prostate Cancer. Cells, 12(1), 124.

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Immunofluorescence Staining of NUP Proteins

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For NPC staining in HEK293T, cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and then incubated for 5 minutes in 0.5% Triton-X-PBS for cell permeabilization. For fixation of hematopoietic cells and NUP98–KDM5A PDX cells, a cytocentrifuge was used to adhere cells to a glass slide by spinning at 400 rpm for 4 minutes. The cells were then rapidly rinsed in 1× PBS–5 mmol/L EGTA, followed by incubation at −20°C in 95% methanol–5 mmol/L EGTA for 30 minutes. The primary antibodies used were mouse anti-NUP107 (Abcam, Mab414, RRID:AB_448181, 1:300) and rat anti-NUP98 (GeneTex, 2H10, RRID: AB_2894964, 1:200). The secondary antibodies used were raised in donkey and conjugated to Alexa Fluor Rhodamine Red-X or Alexa Fluor 647 (Jackson ImmunoResearch; RRID:AB_2340614) at 1:300 diluted in 5% normal donkey serum. Cells were counterstained with DAPI (Invitrogen) diluted in PBS (300 nmol/L) for 2 minutes and then mounted onto glass slides with antifade solution (90% glycerol, 0.5% N-propyl gallate). See Supplementary Methods for additional details.

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Multicolor Imaging of Nuclear Architecture

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To image chromatin distribution, HL60 cells were incubated with Hoechst 33342 dye (Molecular Probes, Eugene, OR) with a final concentration of 5 μg/mL for 20 min before isolation. To stain for nuclear membrane, FM4-64 (Invitrogen, Carlsbad, CA) was added to the isolated nuclei with a final concentration of 100 ng/mL, and incubated for 10 min. Nuclei were then seeded onto poly-lysine-coated coverslips for 10 min before washing. To image nucleoli, cells were stained with NUCLEOLAR-ID Green Detection Reagent (1:1000; Enzo Life Sciences, Farmingdale, NY) according to manufacturer’s instruction, before nuclear isolation. Samples were incubated for half an hour with primary antibodies recognizing nuclear pore complex (Mab414, dilution 1:500; Abcam, Cambridge, UK), lamin A/C (SAB4200236, 1:500; Sigma-Aldrich), HP1α (05-689, 1:500; Millipore), coilin (C1862, 1:500; Sigma-Aldrich), PML (HPA008312, 1:500; Sigma-Aldrich), or RNA pol II (SC-899, 1:500; Santa Cruz Biotechnology, Dallas, TX). The samples were then incubated at 4°C overnight. After washing, samples were incubated for 1 h with an appropriate secondary antibody conjugated with Alexa 488 (1:400; Molecular Probes), and washed again. All stained nuclei were imaged with an LSM700 inverted confocal microscope (40×, NA = 1.1, water immersion; Carl Zeiss). Gain settings were kept the same for all samples of a given experiment.

Chan C.J., Li W., Cojoc G, & Guck J. (2017). Volume Transitions of Isolated Cell Nuclei Induced by Rapid Temperature Increase. Biophysical Journal, 112(6), 1063-1076.

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Nesprin-1 and Nesprin-2 Immunofluorescence and Western Blotting

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Nesprin-1 polyclonal antibodies (SpecII) and mAb K58–398–2 directed against the C-terminus of human Nesprin-1, affinity-purified rabbit anti-Nesprin-1 ABD, and mAb K43–322–2 directed against the N-terminus were used.²² (link) pAbK1 was used to detected Nesprin-2.⁶ (link) Immunofluorescence and western blotting were done as described.²³ (link) Antibodies used were specific for Emerin (4G5, Abcam), LAP-2 (BD Transduction Laboratories), Lamin B1 (Abcam), Lamin A/C (StCruz), SUN1 (Abcam), SUN2 (Abcam), mAb414 recognizing nuclear pore complex (NPC) proteins (Abcam), anti-phospho-Ser139 H2AX (Millipore), anti-phospho-Ser317 Chk1 (Cell Signaling), anti-phospho-Ser345 Chk1 (Cell Signaling), anti-phospho-Thr68 Chk2 (Cell Signaling), Ku70, MSH2, MSH6 (all Abcam), and appropriate secondary antibodies conjugated to Alexa 488/568 (Invitrogen). Imaging was done by confocal laser scanning microscopy (Leica TCS-SP5). Images were processed using TCS-SP5 software.

Sur I., Neumann S, & Noegel A.A. (2014). Nesprin-1 role in DNA damage response. Nucleus, 5(2), 173-191.

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