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Control normal rabbit igg

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Control normal rabbit IgG is a laboratory reagent used as a control in experiments involving antibody-based techniques. It consists of immunoglobulin G (IgG) purified from the serum of normal, non-immunized rabbits. This product serves as a control to help distinguish specific from non-specific binding in assays such as Western blotting, immunoprecipitation, and immunohistochemistry.

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Lab products found in correlation

Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions) Protein g dynabead, by Thermo Fisher Scientific (1 mentions) Anti atf4 antibody, by Cell Signaling Technology (1 mentions) Microchip diapure columns, by Diagenode (1 mentions) Ideal chip qpcr kit, by Diagenode (1 mentions) Igg elution buffer, by Thermo Fisher Scientific (1 mentions) Anti sumo 1 antibody, by Cell Signaling Technology (1 mentions) Dynabeads myone silane, by Thermo Fisher Scientific (1 mentions) Epimark n6 methyladenosine enrichment kit, by New England Biolabs (1 mentions) Dynabeads protein g magnetic beads, by Thermo Fisher Scientific (1 mentions) Rnasin plus rnase inhibitor, by Promega (1 mentions) Rlt buffer, by Qiagen (1 mentions) Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions) Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions) Anti m7g antibody, by MBL Life Science (1 mentions) Protein g sepharose 4 fast flow, by GE Healthcare (1 mentions)

6 protocols using control normal rabbit igg

1

Immunoprecipitation of Chromatin Regulators

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Immunopurified CD8 T cell splenocytes from CD4CreSatb1f/f and CD4CreSatb1f/f mice were stimulated in vitro with CD3-CD28 microbeads for 60 hrs. Cells pellets were resuspended in lysis buffer (50 mM Tris-HCl pH8.0, 1 mM EDTA, 0.5% NP-40, 300 mM NaCl, 10 mM NaF, protease inhibitors, PMSF) and rotated at 4°C for 20 min. The lysate was centrifuged at 12,000g at 4°C for 15 min, and supernatant was used for immunoprecipitation with the indicated antibodies (Satb1, rabbit clone#P472, Cell Signalling; Cdh4, rabbit clone#D8B12, Cell Signalling; Hdac2, Polyclonal, A300-705, Bethyl Laboratories; p66α, Abcam, ab87663; or control normal Rabbit IgG, #2729, Cell Signalling). Proteins were incubated overnight at 4°C and subsequently with Protein G Dynabeads (Life Technologies) for 1 hr. Beads were washed with NETN buffer three times, boiled in Laemmli sampling buffer, and subjected to western blot.
Stephen T.L., Payne K.K., Chaurio R.A., Allegrezza M.J., Zhu H., Perez-Sanz J., Perales-Puchalt A., Nguyen J.M., Vara-Ailor A.E., Eruslanov E.B., Borowsky M.E., Zhang R., Laufer T.M, & Conejo-Garcia J.R. (2017). SATB1 Expression Governs Epigenetic Repression of PD-1 in Tumor-Reactive T Cells. Immunity, 46(1), 51-64.
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2

ATF4 ChIP-qPCR in ER-stressed β-cells

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WT CT215 β-cells were treated with Tm (0.2 μg/mL) for 16 h, followed by cross-linking with 1% (w/v) formaldehyde for 10 min and sonication 5 times for 30 s each at 310 W in an ice water bath with Bioruptor (Sonicbio, Samukawa, Japan). Samples were prepared by iDeal ChIP-qPCR kit (Diagenode, Seraing, Belgium) with anti-ATF4 antibody (Cell Signaling Technology, Danvers, MA, USA) or control normal rabbit IgG (Cell Signaling Technology) according to the manufacturer's instructions. Immunoprecipitated DNA was purified through MicroChIP DiaPure columns (Diagenode) and analyzed by qPCR, as described above.
Kitakaze K., Oyadomari M., Zhang J., Hamada Y., Takenouchi Y., Tsuboi K., Inagaki M., Tachikawa M., Fujitani Y., Okamoto Y, & Oyadomari S. (2021). ATF4-mediated transcriptional regulation protects against β-cell loss during endoplasmic reticulum stress in a mouse model. Molecular Metabolism, 54, 101338.
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3

PROX1 Sumoylation Assay Protocol

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The cells were lysed with the appropriate volume of Pierce™ IP Lysis Buffer (Thermo Fisher Scientific) supplemented with 1X PIC and 1X PMSF and 25 mM of N-ethylmaleimide (NEM) (Thermo Fisher Scientific). Lysates were placed on ice for 10 min and centrifuged at 4 °C for 15 min at 15,000× g to collect protein lysates. For immunoprecipitation, 500 µL of cell lysate (1 mg/mL) was diluted with the Pierce™ IP Lysis Buffer supplemented with PIC, PMSF, and NEM and incubated with PROX1 antibody (Cell Signaling Technology, Danvers, MA, USA) and as the control normal rabbit IgG (Cell Signaling Technology) overnight at 4 °C with rotation. After overnight incubation, 50 mL of protein A/G agarose beads were washed twice with the IP lysis buffer, were added to each sample, and incubated at room temperature for 2 h with gentle rotation, followed by elution with IgG Elution Buffer (ThermoScientific). As described above, the eluted samples were run in 12%, Bis-Tris, 1.0 mm, Mini Protein Gel. The PROX1 SUMOylation was detected by probing the blot with an anti-SUMO1 antibody (Cell Signaling Technology) [17 (link)].
Banerjee P., Kumaravel S., Roy S., Gaddam N., Odeh J., Bayless K.J., Glaser S, & Chakraborty S. (2023). Conjugated Bile Acids Promote Lymphangiogenesis by Modulation of the Reactive Oxygen Species–p90RSK–Vascular Endothelial Growth Factor Receptor 3 Pathway. Cells, 12(4), 526.
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4

N6-Methyladenosine Enrichment Protocol

Cited 1 time
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The EpiMark N6-Methyladenosine Enrichment Kit (item no. E1610S; New England Biolabs, Ipswich, MA) was used to enrich m6A modified RNA per manufacturer’s instructions, with minor modifications. RNA samples were fragmented at 94 °C for 3 min (Barros-Silva et al. 2020 (link)). Dynabeads™ Protein G magnetic beads from Thermo Fisher Scientific (product no. 10003 D) were washed and resuspended in Reaction Buffer. An anti-N6-Methyladenosine (m6A) rabbit monoclonal antibody (product no. 56593, Cell Signaling Technology, Danvers, MA) was bound to the beads, followed by the addition of the fragmented total RNA (3.7 μg) and RNasin ® Plus RNase Inhibitor (product no. N2611; Promega, Madison, WI). The normal Rabbit IgG control (product no. 2729, Cell Signaling Technology), an unconjugated rabbit polyclonal antibody, was used as a nonspecific IgG control for the immunoprecipitation. After the immunoprecipitation, RNA was eluted with Buffer RLT (product no. 79216; Qiagen) then cleaned and concentrated using Dynabeads™ MyOne™ Silane (product no. 37002 D; Life Technologies, Carlsbad, CA). The RNA samples from both input and immunoprecipitated samples were examined by RT-qPCR to quantify m6A enrichment.
Kunovac A., Hathaway Q.A., Pinti M.V., Durr A.J., Taylor A.D., Goldsmith W.T., Garner K.L., Nurkiewicz T.R, & Hollander J.M. (2021). Enhanced antioxidant capacity prevents epitranscriptomic and cardiac alterations in adult offspring gestationally-exposed to ENM. Nanotoxicology, 15(6), 812-831.
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5

m7G tRNA Methylation Profiling Protocol

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m7G tRNA meRIP was performed as previously described(Lin et al., 2018 (link)). Briefly, small RNAs (< 200nt) were purified using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific). Then anti-m7G meRIP was performed on the small RNA by incubating 10 ug small RNAs with 10 ug anti-m7G antibody (MBL International, #RN017M) or normal rabbit IgG control (Cell Signaling, #2229) for 2 hr at 4C. Next, 50 ul pre-washed Protein A/G Magnetic Beads (Thermo Fisher Scientific, #88802) were added to purify the m7G modified small RNAs and incubated for 2 hours at 4C. The beads were washed extensively and the bead bound RNAs were dissociated by boiling the beads in 1X urea loading buffer (Invitrogen) for 5 minutes.
Orellana E.A., Liu Q., Yankova E., Pirouz M., De Braekeleer E., Zhang W., Lim J., Aspris D., Sendinc E., Garyfallos D.A., Gu M., Ali R., Gutierrez A., Mikutis S., Bernardes G.J., Fischer E.S., Bradley A., Vassiliou G.S., Slack F.J., Tzelepis K, & Gregory R.I. (2021). METTL1-mediated m7G modification of Arg-TCT tRNA drives oncogenic transformation. Molecular cell, 81(16), 3323-3338.e14.
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6

Immunoprecipitation and Western Blot

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The mouse retina or cell lysate was incubated overnight at 4 °C with rabbit anti-JNK3 antibody (Cell Signaling Technology) or normal rabbit IgG control (Cell Signaling Technology), followed by incubation with pre-washed protein G-Sepharose 4 Fast Flow (GE Healthcare) for 3 h at 4 °C. The immunoprecipitates were washed efficiently with lysis buffer and analyzed by western blotting. Each experiment was repeated at least three times.
Liu C., Zhang C.W., Zhou Y., Wong W.Q., Lee L.C., Ong W.Y., Yoon S.O., Hong W., Fu X.Y., Soong T.W., Koo E.H., Stanton L.W., Lim K.L., Xiao Z.C, & Dawe G.S. (2017). APP upregulation contributes to retinal ganglion cell degeneration via JNK3. Cell Death and Differentiation, 25(4), 661-676.
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