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Ab1227

Manufactured by Abcam
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Ab1227 is a primary antibody that targets the Protein X. It is a monoclonal antibody produced in mouse and is suitable for use in various immunoassay applications.

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Lab products found in correlation

Biotin hpdp, by Thermo Fisher Scientific (1 mentions) Jetprime transfection kit, by Polyplus Transfection (1 mentions) Immobilon p, by Merck Group (1 mentions) Anti rabbit igg antibody conjugated to horseradish peroxidase, by Jackson ImmunoResearch (1 mentions) Ab6556, by Abcam (1 mentions) Cm5 chip, by GE Healthcare (1 mentions) Biacore t200, by Cytiva (1 mentions) Mabe1095, by Merck Group (1 mentions) Ab6046, by Abcam (1 mentions) Pierce high sensitivity streptavidin hrp antibody, by Thermo Fisher Scientific (1 mentions) Ab82411, by Abcam (1 mentions) A11133, by Thermo Fisher Scientific (1 mentions) Trans blot turbo midi pvdf, by Bio-Rad (1 mentions) Ab9485, by Abcam (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Sc 816, by Santa Cruz Biotechnology (1 mentions) Progesterone 4 pregnene 3 20 dione, by Merck Group (1 mentions) Stat3, by Cell Signaling Technology (1 mentions) Interleukin 6 (il 6), by R&D Systems (1 mentions)

9 protocols using ab1227

1

Recombinant Protein Purification and Antibody Validation

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The purified recombinant human GC1 protein was purchased from Enzo life sciences (ALX-201-177-C010), and the Thioredoxin 1 (Trx1) protein was from Fitzgerald industries (30R-2985). FBS was from VWR (97068-085), the GC1 α antibody was from ThermoFisher (MA517086, Waltham, MA, USA), the GC1 β antibody was from Cayman chemical company (160897), Trx1 antibodies were from cell signaling (2298S and human-specific: 2285S). The caspase 3 antibody was purchased from Novus biologicals (NB100-56708), and the biotin antibody from Abcam (ab1227). The following reagents were purchased from Sigma (St. Louis, MI, USA): DTT (D0632), diamide (D3648), Amicon Ultra-0.5 centrifugal filter (UFC500324), Bradford reagent (B6916), PMSF (P7626), protease inhibitor cocktail (P8340), Etoposide (E1383), and the Caspase 3 activity assay kit (CASP3C-1KT). [α-32P]-GTP was from PerkinElmer (NEG006H250UC), S-nitrosoglutathione from Calbiochem (487920). The jetPRIME transfection kit was from Polyplus transfection (101000015), Diethylamine NONOate (DEA-NO) from Enzo life sciences (ALX-430-034-5005) and biotin-HPDP from ThermoFisher (21341). The Jurkat T cell line, clone E6-1 was purchased from ATCC (TIB-152).
Cui C., Shu P., Sadeghian T., Younis W., Li H, & Beuve A. (2023). Inhibitory Peptide of Soluble Guanylyl Cyclase/Trx1 Interface Blunts the Dual Redox Signaling Functions of the Complex. Antioxidants, 12(4), 906.
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2

Western Blot Analysis of GFP Protein

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Protein samples were separated on 10 % acrylamide gels, and subjected to western blotting on polyvinylidene difluoride membrane (Immobilon-P; Millipore). Blots were blocked with 5 % skim milk in phosphate-buffered saline with Tween (PBST; 7 mM Na2HPO4, 3 mM NaH2PO4, 140 mM NaCl, 5 mM KCl, 0.05 % Tween-20) for 1 h at room temperature. For detection of GFP, blots were incubated with rabbit polyclonal anti-GFP (Abcam ab6556, 1:1000) or anti-biotin (Abcam ab1227, 1:1000) in PBST + 5 % skim milk for 1 h at room temperature, washed with PBST three times for 10 min each at room temperature, incubated with anti-rabbit IgG antibody conjugated to horseradish peroxidase (Jackson Immuno Research 111035003, 1:10,000) for 45 min at room temperature, washed with PBST three times for 10 min each at room temperature, and finally washed once with PBS at room temperature for 10 min. Blots were developed using enhanced chemiluminescent western blotting substrate (Bio-Rad Laboratories).
Waaijers S., Muñoz J., Berends C., Ramalho J.J., Goerdayal S.S., Low T.Y., Zoumaro-Djayoon A.D., Hoffmann M., Koorman T., Tas R.P., Harterink M., Seelk S., Kerver J., Hoogenraad C.C., Bossinger O., Tursun B., van den Heuvel S., Heck A.J, & Boxem M. (2016). A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large. BMC Biology, 14, 66.
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3

Identification of PGK1 as CBR-470-PAP Target

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10 cm dishes of confluent IMR32 cells were exposed to 5 μM CBR-470-PAP with the addition of either DMSO or a 50-fold molar excess of CBR-470-1 (250 μM) for 1 hour at 37 °C. Samples were then UV crosslinked using a Stratalinker 2400 instrument for 10 minutes. RIPA extracted lysates were then fractionated with ammonium sulfate with percent increments of 20. These fractions were then separated via SDS-PAGE and relevant probe-labeling was determined by anti-biotin (1:500, ab1227, Abcam) western blotting as above. A parallel gel was silver stained using the Pierce silver stain kit. Relevant gel slices from the 80 percent fraction were excised and PGK1 identity was determined by LC-MS/MS by the Scripps Center for Metabolomics and Mass Spectrometry. Follow up shRNA knock down studies confirmed PGK1 as the target within this fraction.
Bollong M.J., Lee G., Coukos J.S., Yun H., Zambaldo C., Chang J.W., Chin E.N., Ahmad I., Chatterjee A.K., Lairson L.L., Schultz P.G, & Moellering R.E. (2018). A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling. Nature, 562(7728), 600-604.
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4

Identification of PGK1 as CBR-470-PAP Target

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10 cm dishes of confluent IMR32 cells were exposed to 5 μM CBR-470-PAP with the addition of either DMSO or a 50-fold molar excess of CBR-470-1 (250 μM) for 1 hour at 37 °C. Samples were then UV crosslinked using a Stratalinker 2400 instrument for 10 minutes. RIPA extracted lysates were then fractionated with ammonium sulfate with percent increments of 20. These fractions were then separated via SDS-PAGE and relevant probe-labeling was determined by anti-biotin (1:500, ab1227, Abcam) western blotting as above. A parallel gel was silver stained using the Pierce silver stain kit. Relevant gel slices from the 80 percent fraction were excised and PGK1 identity was determined by LC-MS/MS by the Scripps Center for Metabolomics and Mass Spectrometry. Follow up shRNA knock down studies confirmed PGK1 as the target within this fraction.
Bollong M.J., Lee G., Coukos J.S., Yun H., Zambaldo C., Chang J.W., Chin E.N., Ahmad I., Chatterjee A.K., Lairson L.L., Schultz P.G, & Moellering R.E. (2018). A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling. Nature, 562(7728), 600-604.
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5

SPR Oligonucleotide Binding Kinetics

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Biacore T200 and CM5 chips (GE Healthcare) with cross-linked anti-biotin rabbit polyclonal antibody (Abcam, ab1227, 1:10,000 dilution) were used for all SPR experiments. The instrument was first primed three times with reaction buffer and flow cell 1 (FC1) was used as the reference flow cell, which was unmodified and lacked the oligonucleotide. Flow cell 2 (FC2) was used for the immobilization of the oligonucleotide. The biotin-labeled oligonucleotide was injected over a 1 min period at a flow rate of 5 μL min−1, and immobilization levels of 200–300 RU were routinely observed under these conditions. Protein-DNA binding assays were performed in the reaction buffer at the relatively high flow rate of 10 μL min−1 to avoid or minimize any mass-transport limitation effects. Protein solutions (15, 30, 60, 120, and 240 nM) were injected for 120 s followed by a dissociation in reaction buffer for 60 s. At the end of the dissociation period, the sensor chip was regenerated to remove any remaining bound material by injecting reaction buffer, containing 15 mM NaOH, at 30 μL min−1 for 300 s.
Yu J., Zhu C., Xuan W., An H., Tian Y., Wang B., Chi W., Chen G., Ge Y., Li J., Dai Z., Liu Y., Sun Z., Xu D., Wang C, & Wan J. (2023). Genome-wide association studies identify OsWRKY53 as a key regulator of salt tolerance in rice. Nature Communications, 14, 3550.
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6

Diverse Immunofluorescence and PLA Antibodies

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Primary antibodies used for immunofluorescence and PLA experiments were anti-6-4PP (mouse, NM-DND-002 [Cosmobio] 1/500 dilution), anti-CPD (mouse, NM-DND-001 [cosmobio], 1/200 dilution), anti-XAB2 (mouse, sc-271037 [Santa Cruz Biotechnology], 1/1000 dilution and rabbit, A303-638A [Béthyl], 1/500 dilution), anti-AQR (IPB160 rabbit, A302-547A [Béthyl], 1/500 dilution), anti-CCDC16 (rabbit, HPA027211 [atlas antibodies], 1/250 dilution), anti-PRP19 (rabbit, ab27699 [abcam], 1/500 dilution), anti-DNA:RNA hybrid clone S9.6 (mouse, MABE1095 [Merck Millipore], 1/100 dilution and rabbit, Ab01137-23.0 [Absolute antibody], 1/100 dilution), anti-Biotin (rabbit, ab1227 [abcam] 1/1000 dilution), anti-CSA (rabbit, GTX100145 [genetex], 1/400 dilution), anti- and anti-CSB (mouse, sc398022 [santa-cruz], 1/200 dilution), anti-XPB (rabbit, sc293 [santa-cruz], 1/500 dilution), and anti-XPG (rabbit, sc84663 [santa-cruz], 1/1000 dilution).
Donnio L.M., Cerutti E., Magnani C., Neuillet D., Mari P.O, & Giglia-Mari G. (2022). XAB2 dynamics during DNA damage-dependent transcription inhibition. eLife, 11, e77094.
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7

Biotinylated Protein Quantification in Tissues

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Biotinylated protein levels in mouse tissues were assessed by immunoblotting. Tissue lysates were denatured at 95 °C for 5 minutes in 4X Laemmli sample buffer with 10% β-mercaptoethanol. Samples were loaded on Criterion™ TGX™ any KD gels and run in Novex® Tris-Glycine SDS running buffer. Samples were then transferred to Trans-Blot® Turbo™ Midi PVDF using the Bio-Rad rapid semi-dry system. After Ponceau S staining, membranes were blocked in 2% non-fat milk in Tris-buffered saline with Tween-20® for 1 hour at room temperature. Membranes were incubated overnight at 4 °C with the following primary antibodies at 1:5,000 dilution in blocking solution: Rabbit polyclonal anti-biotin (ab1227, Abcam), rabbit polyclonal anti-transferrin (ab82411, Abcam), rabbit polyclonal anti-BSA (A11133, ThermoFisher Scientific), rabbit polyclonal anti-GAPDH (ab9485, Abcam), rabbit polyclonal anti-beta tubulin (ab6046, Abcam). Goat anti-rabbit HRP secondary antibody or Pierce™ high sensitivity streptavidin-HRP antibody (21130, ThermoFisher Scientific) was added at 1:10,000 dilution in blocking solution and membranes were incubated for 1 hour at room temperature. Signal was visualized using SuperSignal™ West Dura Extended Duration Substrate or SuperSignal™ West Femto Maximum Sensitivity Substrate on HyBlot CL® film.
Ozek C., Krolewski R.C., Buchanan S.M, & Rubin L.L. (2018). Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice. Scientific Reports, 8, 17293.
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8

Antibody Sources and Compound Details

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Antibodies against AR (AR-N20, N terminus, rabbit polyclonal; sc-816, Santa Cruz Biotechnology), PSA (clone ER-PR8, mouse monoclonal; M0750, Dako) and anti-His (H-15; sc-803, Santa Cruz Biotechnology) were from the sources indicated. β-Actin antibody (AC-15, mouse monoclonal; ab6276) and anti-biotin (rabbit polyclonal; ab1227) were from Abcam. STAT3 (mouse monoclonal, 124H6) was from Cell Signaling Technology. Anti-streptavidin (IRDye® 680LT streptavidin, LIC-926-68030) was from LI-COR Biosciences. SINT1 is a natural compound (7 (link)), and LPY19, LPY30, LPY31, and EPI-053 were synthesized (5 (link)). EPI-002 was provided by NAEJA Pharmaceutical Inc. The synthetic androgen R1881 was purchased from PerkinElmer Life Sciences. Enzalutamide (MDV-3100) was purchased from Omega Chem. Bicalutamide was a gift from Dr. Marc Zarenda, AstraZeneca Pharmaceuticals. Interleukin-6 was purchased from R&D Systems (Minneapolis, MN). RPMI 1640 medium was purchased from Life Technologies. All other chemicals including progesterone (4-pregnene-3,20-dione) and dexamethasone were obtained from Sigma-Aldrich unless otherwise stated. PSA (6.1 kb)-luciferase, probasin (PB)-luciferase, PRE-luciferase, GRE-luciferase, 5xGal4UAS-TATA-luciferase, AR(1–558)-Gal4 DNA-binding domain (DBD), AR-YFP, and ARvar567es have been described (4 (link), 8 (link)).
Banuelos C.A., Tavakoli I., Tien A.H., Caley D.P., Mawji N.R., Li Z., Wang J., Yang Y.C., Imamura Y., Yan L., Wen J.G., Andersen R.J, & Sadar M.D. (2016). Sintokamide A Is a Novel Antagonist of Androgen Receptor That Uniquely Binds Activation Function-1 in Its Amino-terminal Domain. The Journal of Biological Chemistry, 291(42), 22231-22243.
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9

Western Blot Protocol for Diverse Protein Detection

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Western blots were performed as described12 (link), using the following primary antibodies: mouse monoclonal anti-beta actin (Abcam, ab8224, RRID: AB_449644, 1:1000 dilution), rat monoclonal anti-tubulin (Abcam, ab6161, RRID: AB_305329, 1:4000 dilution), mouse monoclonal anti-Pgk1 (Abcam, ab113687, RRID: AB_10861977, 1:5000 dilution), rabbit polyclonal anti-biotin (Abcam, ab1227, RRID: AB_298990, 1:3000 dilution), mouse monoclonal anti-c-Myc (clone 9E10, Sigma-Aldrich, M4439, RRID: AB_439694, 1:4000 dilution), mouse monoclonal anti-nuclear pore complex proteins (Mab414, Abcam, ab24609, RRID: AB_448181, 1:5000 dilution). Secondary antibodies used were goat anti-rat IgG (Abcam, ab97057, RRID: AB_10680316, 1:10,000 dilution), donkey anti-rabbit IgG (GE Healthcare, GENA934, RRID: AB_2722659, 1:10,000 dilution) and sheep anti-mouse IgG (GE Healthcare, NA931, RRID: AB_772210, 1:10,000 dilution). See “Quantification and statistical analysis” for a detailed description of western blot quantifications.
Fontana G.A., Hess D., Reinert J.K., Mattarocci S., Falquet B., Klein D., Shore D., Thomä N.H, & Rass U. (2019). Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane. Nature Communications, 10, 2535.
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