When the coverage of cells on cover slips reached about 90%, cells were fixed for 15 minutes at room temperature in 4% paraformaldehyde. After aspiration of fixative, samples were rinsed three times in 1×PBS for 5 minutes each and blocked in Blocking Buffer (1×PBS supplemented with 0.3% Triton X-100 (Sangon Biotech) and 5% normal goat serum (Life Technologies)) for 60 minutes. After incubation with the primary antibody (1:100) overnight at 4℃, samples were rinsed three times in 1×PBS for 5 minutes each and incubated with the anti-rabbit IgG second antibody (1:500) for 60 minutes at room temperature in dark. The normal rabbit IgG (Life Technologies) was used as negative control. The antibodies for immunofluorescence were as follows: rabbit anti-CLDND1 (Abcam), rabbit anti-IgG (Merck Millipore), and anti-rabbit IgG (Alexa Fluor® 488 Conjugate) (Life Technologies). The nuclei was labeled using Hoechst 33258 (Sangon Biotech), and cells were visualized using a fluorescence microscope Ti-S (Nikon, Tokyo, Japan).
Rabbit anti igg
Manufactured by Merck Group
Sourced in United States
Rabbit anti-IgG is a laboratory reagent used for the detection and quantification of immunoglobulin G (IgG) in various biological samples. It is a polyclonal antibody raised in rabbits that specifically binds to IgG molecules. This product can be used in various immunoassay techniques, such as ELISA, Western blotting, and immunohistochemistry, to measure IgG levels in samples.
Automatically generated - may contain errors
Lab products found in correlation
Normal goat serum (ngs), by Thermo Fisher Scientific (2 mentions)
Anti rabbit igg alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions)
Normal rabbit igg, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Sangon (2 mentions)
Anti igg mouse, by Merck Group (2 mentions)
Blocking buffer, by Sangon (1 mentions)
Rabbit anti cldnd1, by Abcam (1 mentions)
Hoechst 33258, by Sangon (1 mentions)
Dm5000b, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Sangon (1 mentions)
Rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Rabbit anti h3k9me3, by Active Motif (1 mentions)
Mouse anti caga, by Santa Cruz Biotechnology (1 mentions)
Anti p erk1 2 t202 y204, by Cell Signaling Technology (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti c jun, by Cell Signaling Technology (1 mentions)
Rabbit anti foxp2, by Cell Signaling Technology (1 mentions)
Magna rip kit, by Merck Group (1 mentions)
Rabbit anti pink1, by Affinity Biosciences (1 mentions)
9 protocols using rabbit anti igg
1
Immunofluorescence Staining of CLDND1
2
Immunofluorescence Assay for HSPA12B
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When the coverage of cells on the coverslips reached approximately 90%, the cells were fixed for 15 min at room temperature in 4% paraformaldehyde. After aspiration of the fixative, the samples were rinsed three times in 1× PBS for 5 min per wash and blocked in Blocking Buffer (1× PBS supplemented with 0.3% Triton X-100 [Sangon Biotech] and 5% normal goat serum [Life Technologies]) for 60 min. After incubation with the primary antibody overnight at 4°C, the samples were rinsed three times in 1× PBS for 5 min per wash and then incubated with the anti-rabbit IgG secondary antibody (1:200) for 60 min at room temperature in the dark. Normal rabbit IgG (Life Technologies) was used as the NC. The antibodies used for the IF assay were as follows: rabbit anti- HSPA12B (Abcam), rabbit anti-IgG (Merck Millipore), and anti-rabbit IgG (Alexa Fluor 488 Conjugate) (Life Technologies). The nuclei were labeled using DAPI (Sangon Biotech), and the cells were visualized using a Ti-S fluorescence microscope (Leica DM 5000B; Leica, Wechsler, Germany).
3
RNA-Binding Protein Immunoprecipitation and ChIP Assays
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RIP assays were performed using the RNA-Binding Protein Immunoprecipitation Kit (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. Five micrograms of rabbit anti-CBX3 (Abcam) antibody and five micrograms of rabbit anti-IgG (Millipore) were used to perform RIP. The enrichment of LINC00998 was determined by qRT-PCR. Chromatin immunoprecipitation (ChIP) assay were conducted according to the manufacturer’s instructions (Invitrogen) using PierceMagnetic ChIP Kit with anti-H3K9me3 antibody. Precipitated DNAs that contained c-Met fragments were then amplified using quantitative qRT-PCR. The primers of c-Met promoter used in ChIP assay were described in Supplementary Table S1 .
4
Histone Demethylase Regulation Assay
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Rabbit anti-KDM4A, anti-ERK1/2, anti-p-ERK1/2 (T202/Y204), anti-c-Jun, and anti-p-c-Jun (S63) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-KDM4B was purchased from Bethyl Laboratories (Montgomery, TX, USA). Rabbit anti-KDM4C was purchased from Novus Biological (Littleton, CO, USA). Rabbit anti-H3K9me3 was purchased from Active Motif (Carlsbad, CA, USA). Rabbit anti-IgG, and anti-p-Tyrosine were purchased from Millipore (Billerica, MA, USA). Mouse anti-CagA, and anti-IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse anti-β-Actin was purchased from Sigma. Lentiviral vector pLKO-control (pLKO), pLKO-shKDM4A (sh4A#1, TRCN0000234912; sh4A#2, TRCN0000234914), -shKDM4B (sh4B#1, TRCN0000018016; sh4B#2, TRCN0000379460), -shKDM4C (sh4C#1, TRCN0000235047; sh4C#2, TRCN0000235048) plasmids were purchased from The RNAi Consortium (TRC).
5
ChIP Assay Protocol for FOXP2 and E-cadherin
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ChIP assays were performed as previously described (11 (link)). The following antibodies were used for immunoprecipitation: rabbit anti-FOXP2 (Cell Signaling Technology #5337, USA), rabbit anti-IgG (Millipore PP64, USA). For immunoprecipitation, 2 μg of each antibody was used. The ChIP DNA sample or 1% total input (5 μl) was used in qPCR with the following primers: E-cadherin upstream -760 bp forward: 5′-CGA GAT CGT GCC ACT GCA CTC-3′ and -610 bp backward: 5′-TGG GCT GAA GCG ATC CTC CTG-3′; E-cadherin upstream -414 bp forward: 5′-AGG AGT TCG AGG CTG CAG TG-3′ and -280 bp backward: 5′-TTC TGA TCC CAG GTC TTA GT-3′; PHF2 upstream -1838 bp forward: 5′-CGT GAA GAA CTG AGC CCA GG-3′ and -1693 bp backward: 5′-CGT GAA GAA CTG AGC CCA GG-3′; PHF2 upstream -1519 bp forward: 5′-GAC AAT GGC TTA AGA GTT AC-3′ and -1403 bp backward: 5′-CTG AAT TCC GGT TGT CAC TG-3′.
6
PINK1 Interactome Identification via RIP
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RIP assays were conducted using the Magna RIP Kit (Millipore, Billerica, MA, USA) [20 (link), 21 (link)]. HCT116 cells (2 × 107/sample) were homogenized with RIP Lysis Buffer, followed by incubation with protein A/G magnetic beads coated with rabbit anti-PINK1 (Affinity) or rabbit anti-IgG (Millipore) overnight at 4 °C. The beads were washed with RIP Wash Buffer six times and incubated with proteinase K buffer to digest proteins. RNAs bound to PINK1 were extracted, purified, and analyzed by real-time PCR.
7
Immunoprecipitation and RNA Extraction
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Cells were lysed with hypotonic lysis buffer (10 mM Tris pH7.5, 10 mM NaCl, 10 mM EDTA, 0.5% TritonX-100, proteinase inhibitor). The lysate was centrifuged and the supernatant was pre-cleaned with rabbit anti-IgG (Millipore). After pre-cleaning, rabbit anti-IgG or rabbit-anti-YB-1 (Abcam, ab12148), together with protein G magnetic beads were added into the samples and incubated at 4 °C for 16-18 h. The magnetic beads were removed and washed with wash buffer (50 mM Tris pH7.5, 150 mM NaCl, 0.05% NP-40). The beads were re-suspended in proteinase K buffer (100 mM NaCl, 10 mM Tris-Cl pH7.0, 1 mM EDTA, 0.5% SDS, proteinase K). Then, RNAs were extracted using the Trizol method.
8
Immunoprecipitation of Protein Complexes
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Harvested cells were lysed for 40 min on ice in IP lysis buffer (0.75% CHAPS, 10% (vol/vol) glycerol, 150 mM NaCl, 50mM Tris pH 7.5) freshly supplemented with protease inhibitors. Supernatants were diluted to adjust the CHAPS concentration to 0.375%. Whole cell lysates (3 to 5 mg total protein) were incubated with anti-IgG rabbit (Millipore, N101), anti-IgG mouse (Millipore, N103), anti-DNA-PKcs (Thermo Fisher, PIMA513244 or Invitrogen, MA5-132238), anti-SIRT2 (Abcam, ab67299) or anti-GFP antibody (Abcam; Ab6556). Protein G beads, A agarose beads (Invitrogen), or FLAG conjugated beads (Sigma A2095) were used to immunoprecipitate antibody bound protein. Complexes were washed 4 times with IP washing buffer (0.375% CHAPS, 10% glycerol, 150 mM NaCl, 50 mM Tris pH 7.5) supplemented with protease inhibitors.
9
ChIP Antibodies for dCTCF and Histone Modifications
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The following antibodies were used for ChIP: dCTCF polyclonal antibodies were generated by New England Peptide by immunizing rabbits with a peptide corresponding to the first 20 aminoacids of dCTCF53 (link). The following antibodies were obtained from commercial sources: anti-H3K4me3 (Abcam #8580), anti-H3K27ac (Abcam #4729), anti-H3K27me3 (Abcam #6002), anti-RNA Pol II pSer2 (Abcam #5095), anti-RNA Pol II pSer5 (Abcam #5408), anti-IgG mouse (Millipore #12-371), and anti-IgG Rabbit (Millipore #12-370).
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