Primary antibodies: PSMA1 (hybridoma kindly provided by Tanaka KG, The University of Tokyo). PSMA4 (Santa Cruz, sc-271297), PSMB7 (Santa Cruz, sc-58410); PSMA1–7 (Enzo, BML-PW8195–0025), PSMB5 (Enzo, bml-pw8895–0025); PSMD6 (Abcam), PSMD11 (GAPDH, ab99414), GAPDH (Abcam, ab8245); IgG1k Isotype Antibody (BioLegend, BLG-400139), 20S core subunits (Enzo, bml-pw8895–0025). Secondary antibodies: Goat α-mouse HRP; Goat α-Rabbit HRP (Jackson labs).
Goat α rabbit hrp
Manufactured by Jackson ImmunoResearch
Sourced in Czechia
Goat α-rabbit-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to rabbit primary antibodies in immunoassays and immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat α mouse hrp, by Jackson ImmunoResearch (8 mentions)
Rat α ha, by Roche (3 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Ibright cl1000 imaging system, by Thermo Fisher Scientific (2 mentions)
Rabbit α hltf a300 230a, by Fortis Life Sciences (2 mentions)
Mouse α ung2, by OriGene (2 mentions)
Goat α lamin b1 sc 6217, by Santa Cruz Biotechnology (2 mentions)
Goat α rat hrp, by Jackson ImmunoResearch (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Benzonase, by Merck Group (2 mentions)
Digitonin, by Merck Group (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Mouse α ha, by BioLegend (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Protein a agarose, by Repligen (1 mentions)
Poly l lysine (pll), by Peptide Institute (1 mentions)
Goat anti rat hrp, by Cell Signaling Technology (1 mentions)
Goat α rat igg cy2, by Jackson ImmunoResearch (1 mentions)
Goat α mouse igg alexa657, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
10 protocols using goat α rabbit hrp
1
Proteasome Subunit Antibody Analysis
2
Immunoblotting Antibodies and Reagents
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Antibodies used for immunoblotting are as follows: Rat α-FLAG L5 (#637303; BioLegend), Rabbit α-HA, Rabbit α-GFP, Rabbit α-Sec62, Rabbit α-Sec63, Rabbit α-Sec61β, Rabbit α-BAG6, and Rabbit α-Sec61α (Chitwood et al., 2018 (link); Mariappan et al., 2010 (link); Snapp et al., 2004 (link)) are a gift from Dr. Ramanujan Hegde (MRC Laboratory of Molecular Biology, Cambridge, UK). Rabbit α-BiP (#11587-1-AP; Proteintech), Mouse α-HA (#901513; Biolegend), Mouse α-PDI (#MA3-018; Affinity Bioreagents), Goat α-Rat-HRP (#7077; Cell Signaling), Goat α-Mouse-HRP (#115-035-003; Jackson ImmunoResearch), Goat α-Rabbit-HRP (#111-035-003; Jackson ImmunoResearch), Goat anti-rat HRP (#7077S; Cell signaling), Goat α-RAT IgG-Cy2 (#112-225-167; Jackson ImmunoResearch), Goat α-Mouse IgG-Alexa657 (#A-21235; Invitrogen). Beads were purchased as follows: Strep-Tactin XT beads (#2-4010-010; IBA), Rat anti-FLAG L5 affinity gel (#651503; Biolegend), Protein A agarose (#CA-PRI-0100; Repligen), Mouse anti-HA magnetic beads (#88837; Pierce), Poly L-lysine (#OKK-3056; Peptides International). Rabbit Reticulocyte Lysate was purchased from Green Hectares (Ph:1-800-GHLYSAT). Detergents were purchased as follows: digitonin (EMD Millipore), Triton X-100 (Thermo Fisher Scientific), sodium deoxycholate (Sigma-Aldrich), SDS (Sigma-Aldrich), and Tween 20 (American Bioanalytical).
3
ADAR1 Expression Evaluation in Transfected 293 Cells
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2 × 106 SA1Q or HA1Q 293 Flp-In T-REx cells were seeded in DMEM/FBS/B/H/10 D. After 24 h, 4 × 105 cells were reverse transfected with the respective amount of snap- or halo-AC C with 2.5 μl Lipofectamine 2000. Doxycycline concentration was kept at 10 ng/ml and after further 24 h cells were lysed in 1× Laemmli (67 mm SDS, 10 mm Tris pH 6.8, 1.1 m glycerol, 0.10 m dithiothreitol, 0.15 mm bromophenol blue) in RIPA Lysis and Extraction Buffer (1% NP-40, 150 mm NaCl, 25 mm Tris⋅HCl pH 7.6, 1% sodium deoxycholate, 0.1% SDS, Thermo Fisher scientific ; supplemented with 1 tablet cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail by Roche per 10 ml). Protein lysates were separated via SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories) . After blocking in 5% dry milk in TBST, the blot was incubated with rabbit α-ADAR1 (1:1000, Bethyl Laboratories A303-884) and rabbit α-GAPDH (1:1000, Cell Signaling #5174) in 5% dry milk-TBST as primary antibodies. As secondary antibody, goat α-rabbit HRP (1:10 000, Jackson ImmunoResearch 111-035-003) in 5% dry milk-TBST was applied. Chemiluminescence was measured with an Odyssey Fc Imaging System (LI-COR ). For additional experimental data as well as further procedural details, see Supporting Information (Supplementary Figure S8 ).
4
Western Blot Analysis of DNA Repair Proteins
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Cells were lysed in Laemmli buffer supplemented with 100 mM dithiothreitol (DTT) and benzonase (1:100, Sigma-Aldrich). Following denaturation at 65°C, samples were separated by SDS-PAGE and transferred to PVDF-membranes (Millipore) by TransBlot semi-dry transfer. Membranes were blocked in 5% (w/v) milk – PBS-T (0.2% Tween-20) and incubated with primary antibody in milk-PBS-T at 4°C overnight followed by incubation with HRP-conjugated secondary antibodies at room temperature (RT). Blots were developed using chemiluminescent substrates (Thermo Scientific) and visualised using either X-ray film or an iBright CL1000 imaging system (Thermo Fisher Scientific). The following antibodies were used for immunoblotting, listed by manufacturer: Primary antibodies: Abcam: Rabbit α-SLF2 (ab122480), rabbit α-VPRBP/DCAF1 (ab202587). Bethyl Laboratories: Rabbit α-HLTF (A300-230A). GeneTex: Rabbit α-SMC6 (GTX116832), rabbit α-NSMCE1 (GTX107136). NIH AIDS Reagent Program: Mouse α-Vif (#6459; (Simon et al., 1995 (link))). Origene: Mouse α-UNG2 (TA503563). Roche: Rat α-HA (11867423001). Santa Cruz: Goat α-Lamin B1 (sc-6217). Sigma-Aldrich: Mouse α-β-actin (A5316), rabbit α-ANKRD32/SLF1 (SAB2701555). Secondary antibodies: Jackson ImmunoResearch: Goat α-mouse-HRP (115-035-146), goat α-rabbit-HRP (115-035-144), and goat α-rat-HRP (115-035-143).
5
Western Blot Analysis of Trypanosome Cytoskeleton
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Whole cells or detergent-extracted cytoskeleton (CSK) samples were prepared as previously described [9 (link)] with a concentration of 1 × 107 or 2 × 107 (BSF or PCF) cells/well, as mentioned according to the experiments. Whole cell (T), CSK-enriched pellets (P), and soluble proteins (S) were prepared as described before [21 (link)], with the addition of 1% NP-40 and 150 mM NaCl. Proteins were separated on SDS-PAGE and transferred onto 0.2 μm PVDF membrane. The following primary antibodies were used in the indicated cases: α-BILBO1 (pAb 1:200), α-TbKINX1B (rabbit polyclonal 1:2000 or mouse monoclonal 1:1000), α-Tubulin (TAT1, 1:1000, [34 (link)]), anti-enolase (1:10,000), and α-His (Sigma H-1029, 1:3000). Incubations with secondary antibodies goat α-rabbit HRP (Jackson 115-055-068) and goat α-mouse HRP (Jackson 115-035-044) were also performed as cited above. Labelling was revealed with Clarity ECL Substrate (Bio-Rad) and revealed using ImageQuant LAS400. Quantifications were performed using ImageJ. Errors bars in graphs represent the standard error (n = 3).
6
Quantitative Western Blotting for hBChE Variants
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Full-length tetrameric WT hBChE was used as a standard for the
determination of protein concentrations of hBChE variants. Serial
dilutions of WT standard hBChE (68 units/mL) were prepared and afforded
final concentrations of 1–10 units/mL. Both the WT hBChE standard
curve and hBChE variant samples were resolved on the same gel by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and then
transferred to nitrocellulose. Western blot analysis included sequential
steps of blocking [PBS and Tween-20 (0.01%) (PBST) with 5% nonfat
dry milk (NFDM), 1 h, 25 °C], primary antibody (i.e., rabbit
anti-hBChE was generously provided by O. Lockridge, 1:1000 in a PBST/NFDM
mixture, 16 h, 4 °C), secondary antibody [i.e., goat α-rabbit
HRP (Jackson ImmunoResearch, West Grove, PA), 1:5000 in a PBST/NFDM
mixture, 1 h, 4 °C], and detection with SuperSignal West Pico
chemiluminescent substrate (Thermo Fisher Scientific). Images were
scanned to a file, and band densitometry analysis was conducted using
ImageJ (National Institutes of Health, Bethesda, MD). Linear regression
analysis of WT hBChE afforded a best-fit line equation. Variant hBChE
protein concentrations were determined using the density of the variant
bands and the best-fit line equation from the WT hBChE standard curve
(data not shown).
determination of protein concentrations of hBChE variants. Serial
dilutions of WT standard hBChE (68 units/mL) were prepared and afforded
final concentrations of 1–10 units/mL. Both the WT hBChE standard
curve and hBChE variant samples were resolved on the same gel by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and then
transferred to nitrocellulose. Western blot analysis included sequential
steps of blocking [PBS and Tween-20 (0.01%) (PBST) with 5% nonfat
dry milk (NFDM), 1 h, 25 °C], primary antibody (i.e., rabbit
anti-hBChE was generously provided by O. Lockridge, 1:1000 in a PBST/NFDM
mixture, 16 h, 4 °C), secondary antibody [i.e., goat α-rabbit
HRP (Jackson ImmunoResearch, West Grove, PA), 1:5000 in a PBST/NFDM
mixture, 1 h, 4 °C], and detection with SuperSignal West Pico
chemiluminescent substrate (Thermo Fisher Scientific). Images were
scanned to a file, and band densitometry analysis was conducted using
ImageJ (National Institutes of Health, Bethesda, MD). Linear regression
analysis of WT hBChE afforded a best-fit line equation. Variant hBChE
protein concentrations were determined using the density of the variant
bands and the best-fit line equation from the WT hBChE standard curve
(data not shown).
7
Chromatin Immunoprecipitation and Western Blot Antibodies
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The following antibodies were used for ChIP: rabbit α-H3K9me3 (Abcam ab8898), rabbit α-H3K36me3 (Abcam ab9050), mouse α-H3K9me2 (Millipore 05-1249), rabbit α-H3 (Abcam ab1719) and mouse α-IgG (Sigma I5381).
For Western blot the following antibodies were used: mouse α-GAPDH (Abcam ab9484), rabbit α-mRFP (Apronex), rat α-HA (Roche), mouse α-GFP (Santa Cruz sc-9996), rabbit α-H3K9me3 (Abcam ab8898), mouse α-tubulin kindly provided by Pavel Draber (Institute of Molecular Genetics of the ASCR, Prague, Czech Republic), goat α-mouse-HRP (Jackson ImmunoResearch), goat α-rabbit-HRP (Jackson ImmunoResearch), goat α-rat-HRP (Santa Cruz sc-2006).
For Western blot the following antibodies were used: mouse α-GAPDH (Abcam ab9484), rabbit α-mRFP (Apronex), rat α-HA (Roche), mouse α-GFP (Santa Cruz sc-9996), rabbit α-H3K9me3 (Abcam ab8898), mouse α-tubulin kindly provided by Pavel Draber (Institute of Molecular Genetics of the ASCR, Prague, Czech Republic), goat α-mouse-HRP (Jackson ImmunoResearch), goat α-rabbit-HRP (Jackson ImmunoResearch), goat α-rat-HRP (Santa Cruz sc-2006).
8
Western Blotting Protein Analysis
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For Western blotting, cells were lysed after 24 h treatment with indicated SFN concentrations in hypotonic extraction buffer (20 mM Tris-HCl pH7.5, 1 mM EDTA, Roche protease inhibitor cocktail) (Figure 5 , Supplementary Figure 4 ) or luciferase assay buffer (Supplementary Figure 5 ). Proteins were separated according to sizes by SDS-PAGE and transferred to a nitrocellulose membrane, which was probed with primary antibodies: m α β-catenin (sc-7963) (SantaCruz), rb α Axin1 (C76H11), rb α Axin2 (76G6), rb α GSK3β (27C10), rb α phospho-GSK3β (D85E12) (CellSignaling), rat α α-tubulin (MCA77G) (Serotec), m α β-actin (A5441), rb α HA (H6908) (Sigma-Aldrich), m α GFP (11814460001) (Roche), rb α RFP (ab62341) (Abcam). The horseradish peroxidase (HRP) activity of secondary antibodies, goat α mouse-HRP and goat α rabbit-HRP (Jackson ImmunoResearch), was detected with a LAS-3000 (FUJIFILM). Intensities of Western blot bands were quantified with AIDA 2D densitometry.
9
Western Blotting of DNA Repair Proteins
10
Western Blot Protein Detection
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Proteins were separated by weight in 12% polyacrylamide-SDS-gels and transferred after to PVDF-membranes (Carl Roth) using a semi-dry transblot system (Carl Roth). Membranes were blocked with 5% skimmed milk powder (Heirler, Radolfzell, Germany) in tris-buffered saline with Tween TBST (0.05% Tween-20, 1 × TBS; 10 × TBS: 250 mM Tris/HCl pH 7.4, 1.5 M NaCl) (blocking solution) for 1 h. Membranes were incubated in primary antibody, dissolved in blocking solution at 4 °C overnight, followed by three washing steps in TBST. Next, a one-hour incubation with the secondary antibody, which was dissolved in blocking solution, and finally, three washing steps with TBST and one wash with 1 × TBS were carried out. Western blots were developed after incubation with the substrate solution (ECL Substrate, BioRad Lab. Inc., Hercules, CA, USA) for 5 min. Antibodies: LRP1 (1:10,000, ab92544, Lot: 6R259330-27, Abcam), MBP (1:1000, MCA409S, Lot: 161031A, BioRad), PDGFRα (1:10,000, sc-338, Lot: E2015, Santa Cruz), α-tubulin (1:10,000, T9026, Lot: 078M4796 V, Sigma), Goat α rabbit HRP (1:5000, 111-035-144, Lot: 132409, Jackson ImmunoResearch Laboratories Inc.), Goat α mouse HRP (1:10,000, 115-035-068, Lot: 132223, Jackson ImmunoResearch Laboratories Inc.), and Goat α rat (1:5000, 112-035-062, Lot: 90553, Jackson ImmunoResearch Laboratories Inc.).
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