Ab9106
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab9106 is an immunohistochemistry primary antibody that detects the presence of a specific target protein in tissue samples. It is a high-quality research tool designed for use in laboratories.
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95 protocols using ab9106
1
Co-immunoprecipitation of Protein Complexes
2
Immunostaining of Meiotic Sz. pombe Cells
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Nuclear spreads of meiotic Sz. pombe cells were prepared as described previously (Loidl and Lorenz 2009 (link)), with the only modification that Lallzyme MMX (Lallemand Inc., Montréal, Canada) at a final concentration of 100 mg/ml was used as the sole enzyme in the spheroplasting solution (Flor-Parra et al. 2014 (link)).
Immunostaining was performed as previously described (Loidl and Lorenz 2009 (link)) using polyclonal rabbit α-myc (ab9106; Abcam PLC, Cambridge, UK) at a 1:500 dilution and monoclonal rat α-GFP [3H9] (ChromoTek GmbH, Planegg-Martinsried, Germany) at a 1:100 dilution as primary antibodies. Antibody-bound protein was visualized using donkey α-rabbit IgG AlexaFluor-555 (ab150062; Abcam) and donkey α-rat IgG AlexaFluor-488 (ab150153; Abcam), both at a 1:500 dilution, as secondary antibodies conjugated to fluorophores. DNA was stained by Hoechst 33342 (Molecular Probes, Eugene, OR, USA) at a final concentration of 1 μg/ml.
Analysis was done using a Zeiss Axio Imager.M2 (Carl Zeiss AG, Oberkochen, Germany) epifluorescence microscope equipped with the appropriate filter sets to detect red, green, and blue fluorescence. Black-and-white images were taken with a Zeiss AxioCam MRm CCD camera controlled by AxioVision 40 software v4.8.2.0. Images were pseudo-colored and overlaid using Adobe Photoshop CC (Adobe Systems Inc., San José, CA, USA).
Immunostaining was performed as previously described (Loidl and Lorenz 2009 (link)) using polyclonal rabbit α-myc (ab9106; Abcam PLC, Cambridge, UK) at a 1:500 dilution and monoclonal rat α-GFP [3H9] (ChromoTek GmbH, Planegg-Martinsried, Germany) at a 1:100 dilution as primary antibodies. Antibody-bound protein was visualized using donkey α-rabbit IgG AlexaFluor-555 (ab150062; Abcam) and donkey α-rat IgG AlexaFluor-488 (ab150153; Abcam), both at a 1:500 dilution, as secondary antibodies conjugated to fluorophores. DNA was stained by Hoechst 33342 (Molecular Probes, Eugene, OR, USA) at a final concentration of 1 μg/ml.
Analysis was done using a Zeiss Axio Imager.M2 (Carl Zeiss AG, Oberkochen, Germany) epifluorescence microscope equipped with the appropriate filter sets to detect red, green, and blue fluorescence. Black-and-white images were taken with a Zeiss AxioCam MRm CCD camera controlled by AxioVision 40 software v4.8.2.0. Images were pseudo-colored and overlaid using Adobe Photoshop CC (Adobe Systems Inc., San José, CA, USA).
3
Comprehensive Antibody Profiling Protocol
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The following antibodies were used: anti-FLAG (Sigma, Cat: F3165, dilution 1:10,000), anti-Myc (Abcam, Ab9106, 1:2500), anti-Phosphothreonine (pThr, Thermo Fisher, 71-8200, 1:2500); anti-Phosphoserine (pSer, Thermo Fisher, 61–8100, 1:2500); anti-LETM1 (Santa Cruz, mouse sc-514136, 1:2500 or Novus Biologicals, rabbit NBP1-33433, 1:10,000). Anti-PINK1 (Abgent, mouse AM6406a, 1:5000 and Novus Biologicals, rabbit BC100–494, 1:5000), anti-Tyrosine hydroxylase (TH, EMD Millipore, AB152, 1:1000), anti-MCU (Cell Signaling, 14997, 1:10,000), anti-NCLX (Abcam, ab136975, 1:5000). Special custom phospho-LETM1 at Thr192 (pT192) polyclonal antibody was generated and purified from a rabbit immunized with carrier protein-conjugated phosphopeptide, CNGHTLpT192RRERR (residues 187–197 of human LETM1, NP_036450.1), using standard protocols by Biogenes (Berlin, Germany, dilution 1:2500).
4
Western Blot Analysis of Cellular Proteins
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Cells were collected and homogenized in radioimmunoprecipitation assay buffer. Total cell protein extracts were quantified and assessed by Western blot as described elsewhere (15 (link)). The following antibodies diluted in blocking solution [phosphate-buffered saline (PBS) and 0.1% Tween 20 containing 5% milk] were used: anti-HSP90 (1:5000; sc13119, Santa Cruz Biotechnology), anti-Flag (1:3000; F7425, Sigma-Aldrich), anti-TMBIM1 (1:1000; MBS1499661, MyBioSource), anti-BAX (1:1000; 06-499, EMD Millipore), anti-MYC (1:3000; ab9106, Abcam), and anti-actin (1:10,000; sc-8432, Santa Cruz Biotechnology). Bound antibodies were detected using peroxidase-coupled secondary antibodies and the enhanced chemiluminescence system.
5
Immunofluorescent Labeling of Phox2B
6
Western Blot Analysis of Protein Extracts
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IRBCs were treated with 0.2% saponin and proteins were extracted using 1.0% Triton-X 100 (w/v) in PBS containing protease inhibitor cocktail (Complete Mini, Roche) at 4 °C for 1 h. The protein fractions were separated by electrophoresis using 5–20% SDS–polyacrylamide gradient gel (ATTO, Tokyo, Japan) in a reducing condition and transferred to polyvinylidene difluoride membranes (Clear Blot Membrane-P, ATTO, Tokyo, Japan). The membrane was probed with rabbit anti-myc polyclonal antibody (1:500; ab9106, Abcam, UK) or rabbit anti-SBP4 polyclonal antibody (1:1000)30 (link) at 4 °C overnight. The membrane was incubated with HRP-conjugated goat anti-rabbit IgG (1:8000; Promega, USA) as the secondary probe. Protein bands were visualized using Immobilon Western Chemiluminescent HRP substrate (Merck Millipore) and detected by ImageQuant LAS 500 (GE healthcare).
7
Immunostaining of Drosophila Brains
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Whole brains were dissected from adult fly heads and transferred into 500 μl ice cold PBS. For fixation the brains were then transferred into 4% (w/v) paraformaldehyde in PBS for 1 h at room temperature. Afterwards the fixative was removed and the brains were blocked with 5% normal goat serum in PBS (15 min). The brains were then infiltrated for 2 h with 6E10 (Aβ1-16, monoclonal, mouse, Covance, 1:500), anti-elav (monoclonal, rat, DHSB, 1:50) or anti-myc antibody (ab9106, polyclonal, rabbit, Abcam, 1:500). All antibodies were diluted in PBT (PBS + 0.05% TritonX-100). Unbound antibodies were removed with three washing steps (500 μl PBT, 10 min each) followed by a 1 h incubation with donkey anti-mouse IgG Alexa Fluor®555, donkey anti-rabbit IgG Alexa Fluor®488 (Molecular Probes, 1:200) or anti-rat IgG TRITC (JacksonImmuno Research, 1:200) as appropriate. To remove unbound secondary antibodies, the brains were washed three times with 500 μl PBT, before they were incubated 10 min with 500 μl Hoechst 33342 dye (1 μg/ml in PBS) to stain the nuclei. Finally, the brains were mounted using VECTASHIELD® mounting medium (Vector laboratory). All steps were performed at room temperature, unless indicated otherwise. All samples were analysed using a Nikon ECLIPSE TE2000-E confocal laser-scanning microscope. Pictures were taken at a magnification of 60 ×.
8
Mammalian Cell Culture and Immunoblotting
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Human Embryonic Kidney (HEK293T) and HeLa tumour cells, as well as mouse neuroblastoma (Neuro2A) and embryonic carcinoma (P19) cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco 10313) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher HYC15-010.02), 2 mM L-glutamine (Gibco 25030), 20 units/ml of penicillin and streptomycin (Gibco 15140) under humidified air containing 5% CO2 at 37 °C. Media for Neuro2A cells was supplemented with 1% Non-essential Amino acids (Gibco 10370-021). Transfections were performed using equal quantities of expression plasmids for each condition. Immunoprecipitation and Western blotting analysis was performed with antibodies to FLAG (F1804, Sigma Aldrich), myc (Ab9106, Abcam), actin (A5441, Sigma Aldrich), β-tubulin (T0198, Sigma Aldrich) together with appropriate fluorescent secondary antibodies, as described48 (link). Immunoblot signals were resolved detected with an Odyssey® infrared imaging system (Li-Cor 9201-02) for analysis.
9
Western Blot Analysis of Cellular Proteins
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Tissue or cellular protein was extracted with 1 × cell lysis buffer (Promega, Madison, WI). Western blot was performed as previously described [9 (link), 11 (link)–15 (link)], with antibodies for HNF4A (ab181604), hexokinase 2 (HK2, ab104836), solute carrier family 2 member 1 (SLC2A1, ab40084), v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN, ab16898), hnRNPU (ab10297), CTCF (ab188408), clusterin (CLU, ab69644), C-X-C motif chemokine receptor 4 (CXCR4, ab124824), trophoblast glycoprotein (TPBG, ab129058), uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA, ab99323), FLAG (ab125243), Myc (ab9106), glutathione S-transferase (GST, ab19256), histone H3 (ab5103), or β-actin (ab6276, Abcam Inc., Cambridge, MA).
10
Whole-Brain Imaging Using Light-Sheet Microscopy
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For whole brain imaging we used a Light-Sheet microscope (Ultra-microscope II, LaVisionBioTec) with fixed lens configuration using a ×4 lens. Images were acquired by an Andor Neo sCMOS camera (2560 × 2160, pixel size 6.5 × 6.5 µm, Andor) in 16 bit. Cleared brains were attached with epoxy glue to the sample holder and imaged at 10-µm steps along the Z axis. Autofluorescence was acquired with a 488-nm laser with 525/50 emission filter. The anti-Myc signal (Alexa-647) was acquired with a 640-nm laser with a 690/50 emission filter. Mice were perfused and brains collected for iDISCO clearing as described by others78 (link). For Myc staining, we used a rabbit anti-Myc antibody (abcam - ab9106) followed by Alexa-647-conjugated Donkey anti-Rabbit secondary antibody (Jaxson immunoResearch, 711-605-152), following manufacturer’s instructions. Nonspecific antibody binding was evident in all brains located mostly around big blood vessels (see Fig. 1c, d ). This antibody noise did not affect the quantification as the Myc signal had high signal-to-noise ratio and labeled nuclei were circular in the 3D analysis. For antibodies concentrations, please see “Immunohistochemistry” in “Methods.”
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