The antibodies and their concentrations used for Western blotting are as follows: anti‐mAcon1 mouse (1 µg/ml; ab83528, Abcam), anti‐ATG8A rabbit (1:1000; ab109364, Abcam), anti‐β‐actin rabbit (1:1000; 4967S, Cell Signaling), anti‐mouse goat IgG‐horseradish peroxidase (HRP) (1:10000; sc2005, Santa Cruz Biotechnology), and anti‐rabbit goat IgG‐HRP (1:10000; sc2004, Santa Cruz Biotechnology) antibodies. The antibodies and their concentrations used for IHC are as follows: anti‐mAcon1 mouse (1:100; ab83528, Abcam), anti‐BRP mouse (1:25; DSHB), anti‐ATG8A rabbit (1:200; ab109364, Abcam), anti‐GFP rabbit (1:1000; Invitrogen), anti‐GFP mouse (1:500; GFP‐G1, DSHB), anti‐mouse goat (1:1000; Alexa Fluor® 488, Invitrogen), anti‐rabbit goat (1:1000; Dylight® 488, Invitrogen), and anti‐rabbit goat (1:1000; Alexa Fluor® 594, Invitrogen) antibodies.
Ab109364
Manufactured by Abcam
Sourced in United Kingdom
Ab109364 is a primary antibody that can be used for the detection of target proteins in various applications such as Western blotting, immunohistochemistry, and immunocytochemistry. It is a polyclonal antibody produced in rabbit and specifically recognizes the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (2 mentions)
Chemidoc system, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dmem media, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions)
Lms 880, by Zeiss (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Ab83528, by Abcam (1 mentions)
Dylight 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Ab14734, by Abcam (1 mentions)
Pxi western blot imager, by Syngene (1 mentions)
A3854, by Merck Group (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Ab6789, by Abcam (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Direct blue 71, by Merck Group (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
9 protocols using ab109364
1
Western Blotting and Immunohistochemistry Antibodies
2
Immunofluorescence of Autophagy Markers
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HeLa cells were grown in DMEM media (Gibco) supplemented with 10% FBS, 2mM L-glutamine, 100 µM penicillin/streptomycin and HEPES. Cells were plated on glass coverslips overnight at 2 × 105 cells/mL and fixed for 20 min in 4% v/v paraformaldehyde in 1xPBS at room temperature, then incubated with 1xPBTx for 10 min. Cells were washed 3 times in 1xPBS and blocked for 30 min with blocking buffer (1% BSA, 1% goat serum in PBS). The blocking buffer was removed and primary antibodies, anti-Sequestosome 1 (SQSTM1/p62) (Abnova H00008878-M01 Mouse; 1:200), anti-GABARAPs (Abcam ab109364 Rabbit; 1:200) or anti-V5 (Abcam 27671 Mouse 1:200), incubated at room temperature for 2 h. Cells were then washed 3 times in 1xPBS. The AlexaFluor-488 (1:200) secondary antibody was incubated at room temperature for 1 h. Cells were then washed 3 times in 1xPBS and counterstained with Hoechst 33342 and mounted in Prolong Gold Antifade. For chloroquine treatment, 200 µM of chloroquine was added for 4 h prior to staining.
3
Immunohistochemical Analysis of Mitochondria and Autophagic Proteins in Fly Thoraxes
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The experiments were carried out as previously described with some modifications (Macchi et al., 2013 (link)). Fly thoraxes were dissected into halves in fixation buffer containing 4% paraformaldehyde and 1% Triton X-100 in PBS and fixed for 20 min at room temperature (RT) without shaking. The specimens were washed with 0.1% Triton X-100 in PBS for 20 min at RT three times. After blocking with 5% normal goat serum (Jackson ImmunoResearch) in 0.1% Triton X-100 in PBS for 2 h at RT, the specimens were stained with primary mouse anti-ATP5A (1:1000, Abcam 14748) and/or primary mouse anti-GABARAP+GABARAPL1+GABARAPL2 antibody (1:500, Abcam ab109364) in blocking buffer overnight at 4°C. After washing with 0.1% Triton X-100 in PBS for 20 min at RT three times, the specimens were stained with secondary antibody (1: 1000 dilutions, anti-mouse IgG Alexa-488, Jackson ImmunoResearch, or Alexa Fluor 647 Phalloidin, Invitrogen A22287) in blocking buffer overnight at 4°C. After washing with 0.1% Triton X-100 in PBS for 20 min at RT three times, the specimens were mounted on the glass slides for imaging. Volumes of 84.2×84.2×5 μm3 were imaged by confocal using LMS880, Zeiss.
4
Protein Extraction and Analysis in Drosophila
5
Autophagy Induction in Aag2 Cells
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To induce autophagy, Aag2 cells were treated with 0.5 µM bafilomycin A1 and 0.5 µM torin-1 for 24 h using 1% DMSO as control. Cells were lysed in passive lysis buffer, and protein concentrations were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Absorbances were read on a GloMax luminometer. Proteins were separated by SDS-PAGE and transferred using the Trans-Blot Turbo Transfer System (Bio-Rad, Oxford, UK). Ae. aegypti ATG8 was detected using a rabbit anti-GABARAP antibody (ab109364; Abcam, Cambridge, UK), and β-actin was detected with a mouse anti-actin antibody (MABT219/JLA20; Merck), both used at 1:1000. Primary antibodies were detected using goat anti-rabbit IgG H&L (HRP) (ab6721; Abcam) and goat anti-mouse IgG H&L (HRP) (ab6789; Abcam) secondary antibodies, respectively. Bands were visualized with ECL reagent (Bio-Rad) on a Bio-Rad ChemiDoc system and Image Lab software (Bio-Rad). Band intensities were analyzed and compared in ImageJ.
6
Recombinant BmAtg8 Protein Expression
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BmAtg8 recombinant protein was expressed in BL21DE3/pET28+BmAtg8 bacteria. The pET28+BmAtg8 expression vector52 was provided by Prof. Yang Cao (South China Agricultural University). The anti-HsGabarap antibody (Abcam, AB109364) was tested against BmAtg8 recombinant protein and protein extracts from silkworm midgut by immunoblotting.
7
Drosophila Protein Extraction and Western Blot
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Triplicate biological cohorts of 3 whole flies per genotype/treatment were homogenized in boiling lysis buffer (50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol), sonicated for 15 seconds, boiled for 10 min, and centrifuged at 13,300 × g at room temperature for 10 min. Samples were electrophoresed on 4–20% gradient gels (Bio-Rad). Western blots were developed using the ChemiDoc system (Bio-Rad). Direct blue staining was used for total protein loading: PVDF membranes were submerged for 5 min in 0.008% Direct Blue 71 (Sigma-Aldrich) in 40% ethanol and 10% acetic acid. PVDF membranes were then rinsed briefly in 40% ethanol and 10% acetic acid solvent, then ultrapure water, air dried, and imaged using the ChemiDoc system. Anti-dAtg8a antibody (ab109364) was from obtained from Abcam. Blots were quantified using ImageLab software (Bio-Rad).
8
Immunofluorescence Microscopy of Drosophila IFMs
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IFMs were prepared from the adult thoraxes dissected in relaxing buffer (0.1 M KCl, 20 mM Tris-HCl, pH = 7.2, 1 mM MgCl2, 1 mM EDTA) [34 (link)]. The specimens were fixed in 4% paraformaldehyde for 30 min. After washing with 0.1% PBST (1× PBS, 0.1% TritonX-100) and subsequent blocking in 10% normal goat serum, the primary antibody diluted with the blocking solution was added and incubated at 4 °C overnight. The following primary antibodies were used: anti-Atg8 antibody (#ab109364, Abcam, Cambridge, UK) (dilution 1/400), anti-Ref(2)P antibody (#ab178440, Abcam) (1/500), anti-ATP5A antibody (#ab14748, Abcam) (1/400), and anti-Cleaved Drosophila Dcp-1 antibody (#9578, Cell Signaling Technology, Inc., Danvers, MA, USA) (1/100).
After washing IFM samples with 0.1% PBST, Alexa fluorescence dye-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) were incubated with the samples for 2 h. For the visualization of F-actin, Alexa Fluor 488-conjugated phalloidin (#A12379, Invitrogen) was added simultaneously. After washing with 0.1% PBST several times, the specimens were embedded with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and observed using a laser scanning confocal microscope (FV10i, Olympus, Tokyo, Japan). Images were acquired at 512 × 512 pixel size. For image processing, FV10-ASW 4.2 Viewer (Olympus) was used.
After washing IFM samples with 0.1% PBST, Alexa fluorescence dye-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) were incubated with the samples for 2 h. For the visualization of F-actin, Alexa Fluor 488-conjugated phalloidin (#A12379, Invitrogen) was added simultaneously. After washing with 0.1% PBST several times, the specimens were embedded with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and observed using a laser scanning confocal microscope (FV10i, Olympus, Tokyo, Japan). Images were acquired at 512 × 512 pixel size. For image processing, FV10-ASW 4.2 Viewer (Olympus) was used.
9
Immunofluorescence, Immunoblotting, and ChIP-seq Protocols
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Antibodies were used for immunofluorescence (IF) or immunoblotting (WB) at the following dilutions. Rabbit polyclonal anti-GFP (A-6455, Molecular Probes), dilution factor 1:5000 (WB); rabbit monoclonal anti-Atg8 (ab109364, Abcam), dilution factor 1:2000 (WB) or 1:100 (IF); mouse monoclonal anti-Flag (F3165, Sigma), dilution factor 1:5000 (WB) or 1:1000 (IF); mouse monoclonal anti-HA (901514, Biolegend), dilution factor 1:1000 (IF); rabbit polyclonal anti-GAPDH (GTX100118, GeneTax), dilution factor 1:10,000 (WB); goat anti-Mouse IgG (H + L) secondary antibody, Alexa Fluor 633 (A-21052, Invitrogen), dilution factor 1:1,000 (IF); donkey anti-Mouse IgG (H + L) Secondary Antibody, Alexa Fluor 555 (A-31570, Invitrogen), dilution factor 1:1,000 (IF); rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (58802, Cell Signaling), dilution factor 1:1000 (WB); mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) (93702, Cell Signaling), dilution factor 1:1000 (WB). For immunoprecipitation, we used a GFP Nanobody/VHH coupled to agarose beads (ChromoTek GFP-Trap Agarose, AB_2631357). For ChIP-seq, we used anti-Flag (Sigma, F3165) and the IgG antibody beads as included in SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9005).
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