Antibodies specific to Cdh1 (DH01), Aurora A, and Plk1 were purchased from Abcam (Cambridge, MA). Antibodies specific to cyclin A2 (sc-751), cyclin (sc-752), cyclin D (sc-753), cyclin E (sc-247), p27 (sc-528), and Cdc20 (sc-8358) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific to α-tubulin (DM1α) were purchased from Sigma-Aldrich. The Skp2 antibody (32-3300), Alexa Fluor 488 anti-mouse antibody (A21202), Alexa Fluor 488 anti-rabbit antibody (A21206), Alexa Fluor 647 anti-mouse antibody (A31571), Alexa Fluor 750 anti-rabbit antibody (A21039), Alex Fluor 680 anti-mouse antibody (A21057), Alexa Fluor 594 rabbit anti-mouse antibody (A11062), Alexa Fluor 488 chicken anti-rat antibody (A21470), Alexa Fluor 594 goat anti-rabbit antibody (A11037), and Alexa Fluor 594 goat anti-chicken (A11039) were purchased from Invitrogen. IRDye 800CW anti-mouse antibody (926-32210) and IRDye 680LT anti-rabbit antibody (926-68021) were purchased from LI-COR Biosciences (Lincoln, NE). Mouse anti-BrdU antibody (347580) was purchased from Becton Dickinson (San Jose, CA). Rat anti-BrdU antibody (OBT0030) was purchased from the Accurate Chemical & Scientific Corporation (Westbury, NY).
Alexa fluor 647 anti mouse antibody
Manufactured by Thermo Fisher Scientific
Alexa Fluor 647 anti-mouse antibody is a fluorescently labeled secondary antibody used for detection in immunoassays. It is reactive against mouse primary antibodies and can be detected using a fluorescence-based readout.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 anti mouse antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 rabbit anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Immobilon fl, by Merck Group (1 mentions)
Mouse anti brdu idu antibody, by BD (1 mentions)
Cy3 anti rat antibody, by Jackson ImmunoResearch (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Mouse anti his antibody, by Merck Group (1 mentions)
4 protocols using alexa fluor 647 anti mouse antibody
1
Antibody Characterization for Cell Cycle Analysis
2
Western Blot Analysis of WRN Protein
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Log phase LCLs (~106 cells/ml) were harvested, washed 1× in PBS, and flash frozen in liquid N2. Cell pellets were lysed in high salt buffer as described previously28 (link), and protein concentrations were measured by the Bradford assay. Approximately 100 μg of total protein were electrophoresed through a NUPAGE 4–12% Bis-Tris polyacrylamide gradient gel (Life Technologies) following the manufacturer’s guidelines. The gel was then transferred overnight to a PVDF membrane (Immobilon-FL, Millipore) using Tris-glycine-methanol transfer buffer. Non-specific membrane interactions were minimized by incubation in a 5% milk solution prepared in TBS-T (Tris-buffered saline, pH 7.5 + 0.05% Tween 20). The membrane was incubated overnight at 4 °C with a WRN- specific mouse monoclonal antibody (clone 195C; Sigma; 1:1000)40 (link) and simultaneously with mouse anti-nucleolin antibody (Invitrogen; 1:1000) to control for loading differences. The membrane was incubated with AlexaFluor 647 anti-mouse antibody (Invitrogen; 1:1000) for 1 hr at room temperature, washed three times in TBS-T, and then imaged on an AlphaInnotech FluorChemQ Imaging Station. Band intensities were quantified using ImageJ software.
3
DNA Fiber Spreads Replication Analysis
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DNA fiber spreads were prepared as described previously (36 (link)). Cells were labeled with 25 μM IdU (5-iodo-2′-deoxyuridine), washed with warm media and then exposed to 50 μM CldU (5-Chloro-2′-deoxyuridine). Cells were lysed with the spreading buffer (200 mM Tris–HCl pH 7.5, 50 mM EDTA and 0.5% SDS) and DNA fiber were stretched onto glass slides. The DNA fibers were denatured with 2.5 M HCl for 1 h, washed with PBS and blocked with 2% BSA in PBS-Tween 20 for 60 min. IdU replication tracts were revealed with a mouse anti-BrdU/IdU antibody (BD Bioscience) and CldU tracts with a rat anti-BrdU/CldU antibody (Abcam). DNA fibers were uniformly labeled with a mouse anti-human single-stranded DNA antibody (Millipore). The secondary antibodies used for the assay were: alexa fluor 488 anti-mouse antibody (Life technologies), alexa fluor 647 anti-mouse antibody (Life technologies) and Cy3 anti-rat antibody (Jackson Immunoresearch). Replication tracts were analyzed with ImageJ software. The probability that two datasets stem from the same distribution was assayed by a non-parametrical Mann–Whitney test (Prism Software).
4
Visfatin Receptor Screening by Microarray
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In order to identify the candidate receptors for visfatin, a binding screen of his-tagged recombinant visfatin against >2500 human membrane proteins, was performed using the Retrogenix Cell Microarray platform (Macclesfield, UK). In brief, binding conditions were optimised for binding of his-tagged recombinant proteins. Expression vectors encoding each of the human membrane proteins were spotted onto glass slides. A HEK293 cell monolayer was cultured over the glass slide, resulting in overexpression of each of the human membrane proteins via reverse transfection. In the primary screen, slides were incubated with 2.5 ug/mL his-tagged visfatin or his-tagged EGF (control), and receptor interactions were detected using a mouse anti-His antibody (Millipore) followed by an AlexaFluor647 anti-mouse antibody (Life Technologies). Receptor hits were identified by visual inspection using ImageQuant software (GE Healthcare, Chicago, IL, USA). Following the primary screen, vectors encoding each of the positive hits were sequenced and 2 confirmatory screens performed. The first confirmatory screen used his-tagged ligands followed by AlexaFluor647 anti-His detection. The second confirmatory screen was performed using his-tagged ligands attached to AlexaFluor647 and anti-His-coated beads (high sensitivity) were used to confirm specificity.
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