Transfected cells were harvested with RIPA buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 0.5% sodium deoxycholate, and 1X protease inhibitor cocktail set III (Calbiochem)) 2 days after transfection. Immunoprecipitation was performed with mouse anti-Myc (9E10, Santa Cruz) antibody or anti-Flag-M2 (F3165, SIGMA) antibody. The antibodies were removed by incubation with protein G Sepharose (Zymed/Invitrogen), and the wash step was repeated 5 times. Proteins were extracted with SDS sample buffer and separated by 10-20% gradient SDS-page (Bio-Rad). To examine the interaction of Flag-C16orf74 with Myc-PPP3CA, we analyzed the immune complexes by western blotting with rabbit anti-Flag and anti-Myc antibodies. To examine the interaction of endogenous C16orf74 with endogenous PPP3CA, Capan-1 cells were harvested in RIPA buffer, and immunoprecipitation was performed with mouse anti-PPP3CA (sc-17808, Santa Cruz) antibody or rabbit anti-C16orf74 (immunized with full-length recombinant C16orf74 protein) antibodies. Then, we collected the antibodies with protein G Sepharose (Zymed/Invitrogen) and repeated the wash steps 5 times. Proteins were extracted with SDS sample buffer and separated by 10-20% gradient SDS-page (Bio-Rad). We analyzed the immune complexes by Western blotting with mouse anti-PPP3CA (sc-17808, Santa Cruz) antibody, or rabbit anti-C16orf74 antibodies.
Mouse anti myc 9e10
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Mouse anti-Myc (9E10) is a monoclonal antibody that specifically recognizes the human c-Myc protein. It is commonly used in various research applications involving the detection and analysis of the c-Myc protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti ha f7, by Santa Cruz Biotechnology (7 mentions)
Mouse anti flag m2, by Merck Group (7 mentions)
Rabbit anti ha y11, by Santa Cruz Biotechnology (4 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Chemiluminescent detection kit, by GE Healthcare (2 mentions)
Calcium phosphate, by Thermo Fisher Scientific (2 mentions)
Mouse anti actin a00702, by GenScript (2 mentions)
Mouse anti α tubulin, by Merck Group (2 mentions)
Mouse anti v5, by Thermo Fisher Scientific (2 mentions)
Gradient sds page, by Bio-Rad (1 mentions)
Sc 17808, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2 f3165, by Merck Group (1 mentions)
Anti cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
Goat anti fibrillarin d 14, by Santa Cruz Biotechnology (1 mentions)
Goat anti gapdh v 18, by Santa Cruz Biotechnology (1 mentions)
Anti phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Anti β actin ac 15, by Merck Group (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Mouse anti gfp, by Roche (1 mentions)
Mouse anti γ tubulin, by Merck Group (1 mentions)
29 protocols using mouse anti myc 9e10
1
Immunoprecipitation of C16orf74-PPP3CA Complex
2
Co-immunoprecipitation Assay for Protein-Protein Interactions
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Cells were lysed in 20 mM Hepes buffer, 10 mM KCl, 1 mM EDTA, 0.2% NP-40, 10% Glycerol44 (link), 59 (link) and co-immunoprecipitation studies were performed as previously described.44 (link) Details can be found in Supplementary Information . The following antibodies were used: mouse monoclonal anti-β-Actin (AC-15, Sigma-Aldrich, St Louis, MO, USA), mouse anti-HSP90 (Heat Shock Protein 90), rabbit polyclonal anti-KLF4 (sc-20691), rabbit polyclonal anti-p21 (C-19; sc-397), mouse monoclonal anti-p53 (DO-1; sc-126), mouse anti-Myc (9E10), mouse monoclonal anti-Caspase-3 (E-8; sc-7272), goat anti-Fibrillarin (D-14; sc-11336), goat anti-GAPDH (V-18; sc-20357), and mouse anti-BRAF wt (sc-5284) (Santa Cruz Biotechnology), rabbit polyclonal anti-BAX, rabbit polyclonal anti-BCL2, rabbit polyclonal anti-E2F1, rabbit polyclonal anti-poly ADP-ribose polymerase, rabbit polyclonal anti-Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), rabbit polyclonal anti-Cleaved Caspase 3 (Asp175) (Cell Signaling Technologies, Danvers, MA, USA). Chemiluminescent detection was used.
3
Cell Lysis and Immunodetection Protocol
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Harvested cells were lysed by vortexing vigorously with glass beads in HB buffer (25 mM MOPS (pH7.2), 5 mM ethylene glycol bis-(2-aminoehylether) tetraacetic acid (EGTA) (pH7.2), 15 mM MgCl2, 150 mM KCl, 50 mM beta-glycerophosphate, 15 mM p-nitrophenylphosphate, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM Na3VO4, 0.2% NP-40, protease inhibitor cocktail (Complete Mini EDTA-free, Roche)). The following antibodies were used to detect target proteins: rabbit anti-HA Y11 (Santa Cruz, Dallas, Texas), mouse anti-GFP (Roche), rabbit anti-GFP (Life Technologies, Carlsbad, California), mouse anti-γ-tubulin (Sigma), rabbit peroxidase anti-peroxidase soluble complex antibody (Sigma, St. Louis, Missouri), and mouse anti-Myc 9E10 (Santa Cruz).
4
Comprehensive Antibody Characterization for Alzheimer's Research
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Reagents were purchased from Sigma-Aldrich unless specified otherwise. Antibodies used in this study were as follow: anti-APP 6E10 (SIG-39320, Covance), anti-Aβ42 12F4 (SIG-39142, Covance), mouse anti-Tau Tau12 (SIG-39416, Covance), mouse anti-Tau HT7 (MN1000, Thermo Scientific Pierce), mouse anti-phosphorylated Tau AT8 (MN1020, Thermo Scientific Pierce), rabbit anti-Tau H150 (SC-5587, Santa Cruz Biotechnology), rabbit anti-Tau (A0024, Dako), rabbit anti-clusterin H330 (SC-8354, Santa Cruz Biotechnology), goat anti-clusterin M18 (SC-6420, Santa Cruz Biotechnology), goat anti-clusterin C18 (SC-6419, Santa Cruz Biotechnology), mouse anti-BIN1 99D (05-449, Millipore), rabbit anti-Gapdh (G9545), rabbit anti-actin (A2066), and mouse anti-Flag (F1804), mouse anti-myc 9E10 (SC-40, Santa Cruz Biotechnology), mouse and goat IgG control (SC-2015 and SC-2028, Santa Cruz Biotechnology), donkey anti-mouse IgG (H+L) IRDye 800 (926-32212, LI-COR Biosciences), donkey anti-rabbit IgG (H+L) IRDye 680 (926-68073, LI-COR Biosciences), donkey anti-rabbit IgG (H+L) IRDye 800 (926-32213, LI-COR Biosciences), and donkey anti-goat IgG (H+L) IRDye 680 (926-68074, LI-COR Biosciences).
5
Immunoprecipitation of KIF1C and Rab6A
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HEK293 cells were transfected with KIF1C and Rab6A constructs 48 hr prior to cell lysis in 50 mM Hepes, pH 7.4, 150 mM NaCl, 1% CHAPS, and protease inhibitors (cOmplete, EDTA free, Roche, Indianapolis, IN). Clarified cell lysate was incubated with llama anti-GFP binding protein (Rothbauer et al., 2008 (link)) conjugated to NHS-activated Sepharose 4 Fast Flow (GE Healthcare Biosciences, Pittsburgh, PA). After washing, the bound fraction was eluted in sample buffer and analyzed by immunoblot with mouse anti-Myc (9E10) or rabbit anti-Rab6A antibody (Santa Cruz Biotechnology, Dallas, TX).
6
Immunostaining and in situ Hybridization Protocols
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Immunostaining and in situ hybridization of imaginal discs were performed according to the standard protocols53 (link), 62 (link). Antibodies were used in this study as follows: rat anti-Ci (2 A) (DSHB, 1:50), mouse anti-Flag (M2) (Sigma,1:200), mouse anti-HA (F7) (Santa Cruz, 1:200), mouse anti-Myc (9E10) (Santa Cruz, 1:5000), mouse anti-β-galactosidase (Sigma, 1:500), mouse anti-En (DSHB, 1:50), mouse anti-Smo (DSHB, 1:50), mouse anti-Cut (DSHB, 1:50), 4,6-iamidino-2-phenylindole dihydrochloide (DAPI) (Santa Cruz, 1:1000). Secondary antibodies used in this study were bought from Jackson ImmunoResearch, and then were diluted at 1:500. For LMB (Sigma) treatment, S2 cells were treated with LMB at a final concentration of 5 nM for 2 h before cells were harvested for mRNA-cap localization assay. For in situ hybridization assay, the primers for mRNA-cap sub-cloning as follows: mRNA-cap upstream 5′-GTTATATGATGCTCATCGAT-3′; mRNA-cap downstream 5′-TTAATTTCCTAATGTCGGGT-3′; the corresponding of cDNA of mRNA-cap was cloned into pBluescript vector. mRNA-cap probe was prepared according to the instruction of the DIG RNA labeling kits (Roche).
7
Immunofluorescence of HEK293T Cells
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Immunofluorescence of HEK293T cells was performed as previously described [27 (link)]. For primary antibodies were used rabbit anti-HA (H6908, Sigma) and mouse anti-myc (9E10, Santa Cruz) and as secondary antibodies were used 594 donkey anti-mouse (Jackson ImmunoReserch) and 594 goat anti-rabbit (Invitrogen). Images were acquired in a Zeiss LSM710 confocal microscope.
8
Antibody-Based Cytoskeleton Analysis
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The following antibodies were used: mouse anti-RhoG (Santa Cruz, sc-1007), mouse anti-myc (9E10) (Santa Cruz, sc-40), mouse anti-vinculin (mouse) (Sigma, V9131), rabbit anti-vinculin (Thermo-Fisher, 700062); rabbit anti-phospho-paxillin (Y118) (Cell Signaling, 2541), rabbit anti-lamellipodin (Cell Signaling, 91138), rabbit anti-Myosin Light Chain and rabbit anti-phospho-Myosin Light Chain (Ser 19) (Cell Signaling, 3671, 3672) mouse anti-alpha tubulin (Sigma, T9026), rabbit anti-tubulin (Abcam, ab18207); Alexa Fluor-488 and Alexa Fluor-594 anti-mouse IgG and anti-rabbit IgG conjugated secondary antibodies and Alexa Fluor-488 and Alexa Fluor-594 Phalloidin (Life Technologies). HRP-conjugated anti-mouse, anti-rabbit and anti-goat secondary antibodies (Jackson Immunoresearch). Hoechst 33342 (AnaSpec Inc., 83218).
Fibronectin (a gift from Keith Burridge, UNC-Chapel Hill, Chapel Hill, NC) and collagen type I (Thermo Fisher, A1048301) were used at indicated concentrations to coat coverslips. The contractility inhibitors (−)-blebbistatin (EMD Millipore) and Y27632 (LC Laboratories) were used as indicated. Nocodazole (Sigma) was used as indicated below.
Fibronectin (a gift from Keith Burridge, UNC-Chapel Hill, Chapel Hill, NC) and collagen type I (Thermo Fisher, A1048301) were used at indicated concentrations to coat coverslips. The contractility inhibitors (−)-blebbistatin (EMD Millipore) and Y27632 (LC Laboratories) were used as indicated. Nocodazole (Sigma) was used as indicated below.
9
Immunoprecipitation of Cell Lysates
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Cells were washed three times with PBS and lysed with ice-cold 0.1% NP40 lysis buffer (50 mM Tris-HCl [pH 7.5], 0.1% NP40, 150 mM NaCl, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and protease inhibitors). All subsequent steps were performed at 4 °C. Cell lysates were pre-cleared with 50 µL of Sepharose CL-4B beads (Sigma Aldrich) for 30 min, and 1 mg of total proteins were immunoprecipitated with 1 µg of the indicated antibodies (rabbit or goat anti-TP53 [FL393, Santa Cruz Biotechnology], mouse anti-FLAG [M2, Sigma Aldrich], mouse anti-MYC [9E10, Santa Cruz Biotechnology], or mouse anti-BCAR1 [21/p130[Cas], BD Biosciences]) for 16 h. After incubating the lysates with 10 µL of 50% Protein A/G agarose beads slurry (Thermo Fisher Scientific) for 2 h, the agarose beads were washed three times using ice-cold 0.1% NP40 lysis buffer before being subjected to SDS-polyacrylamide gel electrophoresis and protein analysis.
10
Comprehensive Western Blotting Protocol
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3–5 × 106 cells were collected per sample, resuspended in 6x Laemmli Buffer and heated at 95 °C for 10 min. The samples were subjected to SDS-PAGE gel electrophoresis using 10% polyacrylamide gels. The gels were then stained with SERVA blue G or blotted on a 0.45 µm nitrocellulose blotting membrane (Neolabs). To verify the protein transfer, the membrane was stained with Ponceau S (SERVA). The membrane was blocked with 5% milk in TBS-Tween and incubated with appropriate concentrations of first and secondary antibodies. Western Lightning Ultra (Perkin Elmer) was used as a chemiluminescence system and signals were detected with the LAS-4000 imager (GE Healthcare) and CCD camera (Fujifilm). Antibodies used were: rabbit anti-Aldolase (1:50000) (Clayton, 1987 (link)); mouse anti-myc 9E10 (Santa Cruz, 1:200); rabbit Peroxidase anti-Peroxidase (Sigma, 1:20000); rat anti-ribosomal protein S9 (1:1000); anti-Trypanothione Reductase (rabbit, gift from L. Krauth-Siegel, BZH Heidelberg); mouse anti-V5 (Biorad, 1:2000); anti-SCD6 and anti-DHH1 (from S. Kramer, University of Wurzburg, 1:10000 and 1:15000 respectively) and rabbit anti-BiP (from J. Bangs, University of Buffalo, 1:1000).
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