Normal rabbit igg

Manufactured by Cell Signaling Technology

Sourced in United States, China, United Kingdom, Japan

Normal rabbit IgG is a control antibody purified from normal rabbit serum. It can be used as a negative control in various immunoassays and other applications where a non-specific rabbit antibody is required.

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Close spelling variants of this product

Normal rabbit IgG antibody (26 citations) Normal IgG (24 citations) Normal rabbit IgG (#2729) (15 citations) Anti-normal rabbit IgG (14 citations) IgG rabbit (9 citations) Control normal rabbit IgG (6 citations) Normal rabbit/mouse IgG (4 citations) Normal rabbit immunoglobulin G (IgG) (3 citations) Anti-Matrin3 or normal rabbit IgG (2 citations) Normal Rabbit IgG isotype control (2 citations)

310 protocols using normal rabbit igg

Sequential Chromatin Immunoprecipitation

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S2 cells were double crosslinked, sonicated, and ChIP’ed as above, using 5 μL anti-Atro sera (SG2524), and 10 μL normal rabbit IgG (Cell Signaling Technology). After the first ChIP, beads were washed and eluted for re-ChIP as described in Truax and Greer (2012) (link). Eluted chromatin was incubated in BSA-blocked Dynabeads for 2 hr to remove any leftover antibodies. The supernatant containing eluted chromatin was re-ChIP’ed by incubating in beads conjugated with the appropriate amount of antibodies overnight (5 μL anti-Atro sera (SG2524), 10 μL anti-Trl sera (gift from K. White), 10 μL normal rabbit IgG (Cell Signaling Technology)). After the re-ChIP, the beads were washed and eluted as normal double crosslink ChIP samples above. qPCRs were done in technical triplicates with SYBR green PCR Master Mix (Applied Biosystems, Canada, 20 μL reaction volume; qPCR was performed three times for each ChIP samples). Percent input and errors were calculated using standard percent input calculations. qPCR primer pairs are listed in Supplementary file 2.

Yeung K., Boija A., Karlsson E., Holmqvist P.H., Tsatskis Y., Nisoli I., Yap D., Lorzadeh A., Moksa M., Hirst M., Aparicio S., Fanto M., Stenberg P., Mannervik M, & McNeill H. (2017). Atrophin controls developmental signaling pathways via interactions with Trithorax-like. eLife, 6, e23084.

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Immunoprecipitation of HIF-2α in HCC cells

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Hypoxia‐inducible factor‐2α protein was immunoprecipitated from a 35‐mm plate of HCC cells. Whole‐cell extracts were incubated with protein A beads (16‐156; Millipore) and normal rabbit IgG (2729S; Cell Signaling Technology) to clear non‐specific binding. We then incubated the supernatants with 2 μL anti‐HIF‐2α (NB100‐122; Novus Biologicals) antibody or normal rabbit IgG (2729S; Cell Signaling Technology) for 3 h before mixing with protein A beads. After binding overnight, the supernatant was removed by centrifugation, and the immunoprecipitated proteins were eluted and detected by WB.

Sun L., Kong Y., Cao M., Zhou H., Li H., Cui Y., Fang F., Zhang W., Li J., Zhu X., Li Q., Song T, & Zhang T. (2017). Decreased expression of acetyl‐CoA synthase 2 promotes metastasis and predicts poor prognosis in hepatocellular carcinoma. Cancer Science, 108(7), 1338-1346.

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Protein Interaction and ncRNA Analysis

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RIP and dilncRNA analysis was carried out in NIH2/4 as previously described^{16 (link)}, with minor modifications. Briefly, I-SceI-GR-expressing NIH2/4 cells were transfected with the plasmid encoding for N-protein or with an EV as a control at 24 h before triamcinolone acetonide 0.1 µM (Sigma-Aldrich) administration. IP was performed using 5 μg of anti-N-protein, or 10 μg of anti-53BP1 (Supplementary Table 1), or with normal rabbit IgGs (Cell Signaling) as a mock IP.

Gioia U., Tavella S., Martínez-Orellana P., Cicio G., Colliva A., Ceccon M., Cabrini M., Henriques A.C., Fumagalli V., Paldino A., Presot E., Rajasekharan S., Iacomino N., Pisati F., Matti V., Sepe S., Conte M.I., Barozzi S., Lavagnino Z., Carletti T., Volpe M.C., Cavalcante P., Iannacone M., Rampazzo C., Bussani R., Tripodo C., Zacchigna S., Marcello A, & d’Adda di Fagagna F. (2023). SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence. Nature Cell Biology, 25(4), 550-564.

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Immunoprecipitation of SARS-CoV-2 N-Protein

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Huh7 were transfected with the plasmid encoding for N-protein^{40 (link)} or with an EV as control. Forty-eight hours post-transfection, cells were irradiated or not. One hour post-IR, cells were collected by trypsinization and washed twice in ice-cold 1× PBS. Cell pellets were lysed in IP buffer supplemented with 1× protease inhibitors (Roche), 0.5 mM dithiothreitol, 40 U ml⁻¹ RNaseOUT and 1,000 U ml⁻¹ DNase I (Roche), and incubated at room temperature for 15 min and at 4 °C for an extra 15 min with gentle rotation. Lysates were cleared by centrifugation at maximum speed for 20 min at 4 °C. After addition of 5 mM EDTA (pH 8), lysates were incubated overnight at 4 °C with 5 μg of anti-N-protein (40588-T62 Sino Biological) or with normal rabbit IgGs (Cell Signaling), which were previously bound to Dynabeads Protein G (ThermoFisher). After five washes with 1 ml of IP buffer, immunoprecipitated proteins were analysed by immunoblotting.

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Chromatin Immunoprecipitation Assay Protocol

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ChIP assays were performed using the “Simple ChIP Plus Enzymatic Chromatin IP Kit” (Cell Signaling) according to the protocol described. Briefly, MDA-MB-231 cells were transfected with siNS or siHP1γ when specified and were subjected to hormonal treatment (100 nM Dex) for 2 h. Cells were then cross-linked using 1% formaldehyde (Sigma-Aldrich) in 15 cm in diameter culture dishes containing 20 ml medium and incubated for 10 min at RT. Then, 2 ml of 10X glycine (Cell signaling) was added to each 15 cm in diameter dish and cells were further incubated for 5 min at RT to stop the cross-linking. Cell extracts were then prepared, and chromatin digested and sonicated. Immunoprecipitation of sonicated chromatin solutions was conducted overnight at 4°C with the following antibodies: normal rabbit IgG (#2729; Cell Signaling Technology), anti-GR/D6H2L (#12041; Cell Signaling Technology), anti-HP1γ (ab10480; Abcam), anti-PRMT5 (07-405; Sigma-Aldrich), and p-S2/S5 RNA pol II (#4735; Cell signaling Technology). Cross-linking was reversed by heating, and immunoprecipitated DNA was purified and analyzed by qPCR as described above. Results are expressed relative to the signal obtained from input chromatin. Primer sequences are indicated in Table 2.

Noureddine L.M., Ablain J., Surmieliova-Garnès A., Jacquemetton J., Pham T.H., Marangoni E., Schnitzler A., Bieche I., Badran B., Trédan O., Hussein N., Le Romancer M, & Poulard C. (2023). PRMT5 triggers glucocorticoid-induced cell migration in triple-negative breast cancer. Life Science Alliance, 6(10), e202302009.

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Immunoprecipitation and Western Blot Analysis of RAB27B

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The cells were lysed in 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 5 mM EGTA (pH 8.0), 50 mM NaF (pH 8.0), 10% glycerol, 1.5 mM MgCl₂, and 1% Triton X-100, and the protein concentration was determined by the BCA method. One milligram of cell lysate was pretreated with protein A hand G magnetic beads (Millipore Corp., Bedford, MA, USA) and incubated with 5 µg anti-DDK antibody (AB205606, Abcam) or normal rabbit IgG (# 2729, Cell Signaling). The complex was incubated with protein Ahamg magnetic beads for 2 h. The immune complex was washed, eluted and denatured in Laemmli buffer. The immuno-precipitated proteins were transferred to nitrocellulose membranes after SDS gel electrophoresis. For immunoblotting, the primary antibody used was anti-RAB27B (# 44813, Cell Signaling).

Liu M., Li Y., Zhang C, & Zhang Q. (2022). Role of aurora kinase B in regulating resistance to paclitaxel in breast cancer cells. Human Cell, 35(2), 678-693.

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ChIP-qPCR Protocol for Histone Modifications

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For ChIP experiments, cells were cross-linked with formaldehyde before chromatin was extracted, sonicated, and incubated with primary antibodies Rabbit Monoclonal anti-Histone H3 (acetyl K27) (Abcam, Cambridge, UK) (#ab4729, 1:500); Mouse monoclonal ANTI-FLAG® M2 antibody (Merck Millipore, Burlington, MA) (#F1804, 1:100); Rabbit Polyclonal anti-BRD4 (Bethyl, Montgomery, TX) (#A301-985A100, 1:100); Mouse Monoclonal anti-KAT3B/p300 (Abcam, Cambridge, UK) (#ab14984, 1:100); Normal mouse IgG (Santa Cruz, Santa Cruz, CA) (#sc-2025, 1:200); Normal rabbit IgG (Cell Signaling Tech, Danvers, MA) (#2729, 1:200) overnight. Antibody complexes were then captured with Dynabeads Protein G (Thermo Fisher, 10004D), and DNA was eluted, de-crosslinked, and purified. ChIP signal were calculated by qPCR (Supplementary Table 4) relative to input levels after (IgG) background subtraction.

Yu D., Liang Y., Kim C., Jaganathan A., Ji D., Han X., Yang X., Jia Y., Gu R., Wang C., Zhang Q., Cheung K.L., Zhou M.M, & Zeng L. (2023). Structural mechanism of BRD4-NUT and p300 bipartite interaction in propagating aberrant gene transcription in chromatin in NUT carcinoma. Nature Communications, 14, 378.

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ChIP-qPCR Analysis of MYC Binding

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DU145 cells were fixed by addition of 37% formaldehyde to a final concentration of 1% formaldehyde and incubated at room temperature for 10 min. Crosslinking was stopped by the addition of glycine to a final concentration of 0.125 M. Cells were then scraped, and samples were prepared using SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling, #9003) according to the manufacturer’s protocol. The chromatin fractions were incubated in each case with 10 mg of antibodies to one of the following: MYC (Cell Signaling, #13987), human RPL30 or normal rabbit IgG (both provided by Cell Signaling kit, Cell Signaling) at 4°C overnight with Magnetic Protein G Beads. After extensive washing and final elution, the product was treated at 65°C overnight to reverse the crosslinking. Input DNA and immunoprecipitated DNA were purified using a kit column and analyzed by qPCR using SYBR Green Supermix (Bio-Rad) with the following sets of primers (both proximal and distal promoter regions): human WWP1 promoter (forward, 5′-GTCCGGAGTTGGAGGCTTT-3′; reverse, 5′-GACCCCACACCTCCCTTC-3′), human JunB (forward, 5′-AAGCCCACAGAGAGAGGTGGAAG-3′; reverse, 5′-CCAGAAGGTGGTGCCTTTTTATTG-3′). All results were normalized to the respective input values.

Lee Y.R., Chen M., Lee J.D., Zhang J., Lin S.Y., Fu T.M., Chen H., Ishikawa T., Chiang S.Y., Katon J., Zhang Y., Shulga Y.V., Bester A.C., Fung J., Monteleone E., Wan L., Shen C., Hsu C.H., Papa A., Clohessy J.G., Teruya-Feldstein J., Jain S., Wu H., Matesic L., Chen R.H., Wei W, & Pandolfi P.P. (2019). Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway. Science (New York, N.Y.), 364(6441), eaau0159.

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Immunoprecipitation of USP7 and TRIM27

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Immunoprecipitations (IP) were performed as before (Li et al., 2012 (link)). Briefly, treated or untreated 2D10 cells were washed and lysed in 150 mM NaCl, 50 mM Tris-HCl (pH 7.2), 0.5% NP-40, and protease inhibitor cocktail (Cell Signaling) followed by an incubation at 4°C for 20 min with vortex. Clarified lysates were precleared with protein A/G agarose beads (Thermo Scientific), followed by IP with either anti-USP7 monoclonal antibody (Bethyl), anti-TRIM27 polyclonal antibody (MyBioSource), or normal rabbit IgG (Cell Signaling). IP was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with 1^st antibodies of anti-USP7 or anti-Trim27 antibody and 2^nd antibodies of horseradish peroxidase-conjugated secondary antibody, followed by chemiluminescence detection (Amersham).

Li D., Dewey M.G., Wang L., Falcinelli S.D., Wong L.M., Tang Y., Browne E.P., Chen X., Archin N.M., Margolis D.M, & Jiang G. (2021). Crotonylation sensitizes IAPi-induced disruption of latent HIV by enhancing p100 cleavage into p52. iScience, 25(1), 103649.

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ATF4-Bound Chromatin Profiling

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Two million neurons were collected and subjected to chromatin IP with antibodies to ATF4 (C-20, Santa Cruz), or normal Rabbit IgG (Cell Signaling) as a control following procedures as described^{41 (link)}. Specific primers for gene promoter regions (Supplementary Table 1b) were used in qPCR to detect the enrichment of specific DNA sequences in the ATF4 bound chromatin fraction.

Wang X., Ye F., Wen Z., Guo Z., Yu C., Huang W.K., Ringeling F.R., Su Y., Zheng W., Zhou G., Christian K.M., Song H., Zhang M, & Ming G.L. (2019). Structural interaction between DISC1 and ATF4 underlying transcriptional and synaptic dysregulation in an iPSC model of mental disorders. Molecular psychiatry, 26(4), 1346-1360.

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