Rabbit igg

Manufactured by Proteintech

Sourced in United States, China

Rabbit IgG is a purified immunoglobulin fraction isolated from rabbit serum. It is a commonly used secondary antibody in immunological techniques such as Western blotting, ELISA, and immunohistochemistry.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Southern Biotech (3 mentions) Qnano platform, by Izon Science (3 mentions) Rabbit anti hnrnpa1 antibody, by Cell Signaling Technology (3 mentions) β actin, by Proteintech (3 mentions) Ez magna rip rna binding protein immunoprecipitation kit, by Merck Group (2 mentions) Mirvana paris kit, by Thermo Fisher Scientific (2 mentions) Anti flag antibody, by Cell Signaling Technology (2 mentions) Ip lysis buffer, by Thermo Fisher Scientific (2 mentions) Mouse igg, by Proteintech (2 mentions) Anti flag, by Merck Group (2 mentions) Rip rna binding protein immunoprecipitation kit, by Merck Group (2 mentions) Anti gst, by Proteintech (2 mentions) Anti g6pd, by Proteintech (2 mentions) Rabbit igg, by Abcam (2 mentions) Ht7700, by Hitachi (2 mentions) Ultravision block, by Thermo Fisher Scientific (1 mentions) Hrp polymer, by Thermo Fisher Scientific (1 mentions) Antibodies against gapdh, by Bioworld Technology (1 mentions) Ha tag, by Proteintech (1 mentions) His tag, by Proteintech (1 mentions)

34 protocols using rabbit igg

Isolation and Characterization of ADSC-Derived Exosomes

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Supernatant was collected after culturing ADSCs in Dulbecco’s modified Eagle’s medium and nutrient mixture F-12 medium for 48 h. The supernatant was subjected to differential centrifugation at 300 × g for 5 min, 2000 × g for 15 min and 10 000 × g for 35 min. Then, the supernatant was filtered through a 0.22-μm filter (Millipore, MA, USA). Finally, the supernatant was transferred to a 100,000 molecular weight cut off (MWCO) ultrafiltration tube (Sartorius, Hamburg, Germany) and centrifuged at 3000 × g for 1.5 h. The supernatant was removed and the pellet was resuspended as ADSC-Exos with an appropriate amount of phosphate-buffered saline (PBS).
Exosome morphology was examined via transmission electron microscopy (TEM). Particle size distribution was determined via nanoparticle tracking analysis ( Particle Metrix, Germany). The presence of exosomal markers, including alix (rabbit IgG, Proteintech, Wuhan, China), CD9 (rabbit IgG, Proteintech), CD63 (rabbit IgG, Proteintech) and calnexin (rabbit IgG, Proteintech), was confirmed via western blot analysis as described below.

Ren H., Su P., Zhao F., Zhang Q., Huang X., He C., Wu Q., Wang Z., Ma J, & Wang Z. (2024). Adipose mesenchymal stem cell-derived exosomes promote skin wound healing in diabetic mice by regulating epidermal autophagy. Burns & Trauma, 12, tkae001.

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Characterization and Uptake of Exosomes

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Transmission electron microscopy (TEM; HT7700, Hitachi, Tokyo, Japan) was performed for morphological analysis of isolated exosomes. The absolute size distribution of exosomes was determined using the qNano platform (iZON® Science, Christchurch, New Zealand). Western blotting of proteins (CD9, CD63, TSG101, and GM130) in exosomes was conducted as described previously [23 (link)] with the following primary antibodies: CD9 (1:1000; rabbit IgG, Proteintech, Rosemont, IL, USA), CD63 (1:1000; rabbit IgG, Proteintech), TSG101 (1:1000; rabbit IgG, Proteintech), and GM130 (1:500; rabbit IgG, Abcam, Cambridge, UK). Exosomes were then labeled with green fluorescent dye (DIO; Life Technologies, Carlsbad, CA, USA), and excess dye was removed by ultracentrifugation at 110,000×g for 70 min at 4 °C. Exosome pellets were washed three times and resuspended in PBS. HUVECs were incubated with DIO-labeled exosomes for 8 h, and cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Southern Biotech, Birmingham, AL, USA); uptake was observed by fluorescence microscopy.

Liang B., Liang J.M., Ding J.N., Xu J., Xu J.G, & Chai Y.M. (2019). Dimethyloxaloylglycine-stimulated human bone marrow mesenchymal stem cell-derived exosomes enhance bone regeneration through angiogenesis by targeting the AKT/mTOR pathway. Stem Cell Research & Therapy, 10, 335.

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RIP-qPCR Analysis of miRNA Binding

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RIP was conducted using EZ-Magna RIP RNA binding protein immunoprecipitation kit (Millipore). Brie y, the cells were collected and lysed in the frozen lysis buffer supplemented with protease inhibitor, RNase inhibitor and 1 mM phenylmethylsulfonyl uoride. The lysis buffer was centrifuged at 14000 g for 15 min, and 50°L lysis buffer was stored as input. The protein extract (1 mg) was incubated with rabbit IgG (Proteintech, Rosemont, IL, USA) at 4℃ overnight and then treated with 30°L A/G protein magnetic beads at 4 ℃ for 4 h. Afterwards, the beads were washed 5 times, and the miR of co-immunoprecipitation was extracted using mirVana PARIS kit (Ambion, Austin, Texas, USA). The extracted miR was reverse transcribed and analyzed by real-time PCR. In addition, miR folding enrichment in immunoprecipitation samples was presented in the form of percentage input, with IgG as isotype control.

Zheng Y., Zhu K., & Wang G. (2021). Mechanism of miR-590-3p carried by tumor-derived extracellular vesicles in promoting invasion and metastasis of ovarian cancer.

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Immunohistochemical Analysis of CD24 Expression

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Tissues were fixed overnight in 4% paraformaldehyde in PBS (pH 7.4) at 4 °C. Fixed tissues were embedded in paraffin and sliced. Sections were prepared for staining first by deparaffinization followed by hydration in the following solutions: 3 washes of xylene 5 min each, two washes of 100% ethanol 10 min each, two washes of 95% ethanol 10 min each and two washes in distilled water 5 min each. Antigen retrieval was obtained by incubation with a heated citrate buffer (sodium citrate 10 mM, pH 6) for 15 min. Immunohistochemistry was performed as per our standard procedures. Briefly, after antigen retrieval sections were incubated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. Non-specific background staining was blocked by incubating in UltraVision Block (Thermo Scientific, # TA-060-PBQ) for 5 min at room temperature. CD24 staining was done by incubating in rabbit anti-CD24 mAb Anti-CD24 mAb Rabbit (Proteintech, St. LeonRot, Germany GMBH, Cat No. 10600-1-AP) at a dilution of 1: 400 overnight at 4° C. For isotype control staining Rabbit IgG (Proteintech, St. LeonRot, Germany GMBH, Cat No. 30000-0-AP) was used. Detection was achieved using HRP Polymer (Thermo Scientific, # TL-060-PH) followed by incubation with peroxidase compatible DAB chromogen.

Vasileva M.H., Bennemann A., Zachmann K., Schön M.P., Frank J, & Ulaganathan V.K. (2024). CD24 flags anastasis in melanoma cells. Apoptosis : an international journal on programmed cell death.

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Western Blot Antibody Validation

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Antibodies against OTUD5 (#21002-1-AP), FLAG-Tag (#20543-1-AP), HA-Tag (#51064-2-AP), His-Tag (#66005-1-IG), Rabbit IgG (#30000-0-AP), Mouse IgG (#B900620) were purchased from Proteintech (Wuhan, China). Antibodies against ERK (#sc-514302), P-ERK (#sc-7383), JNK (#sc-7345), and P-JNK (#sc-6254) were purchased from Santa Cruz (CA, USA). Antibodies against TAK1 (#5206S), P-TAK1 (Ser412) (#9339S), TAB2 (#3745S), Ubiquitin (P4D1) (#3936), P38 (#8690S) and P-P38 (#4631S) were purchased from Cell Signaling Technology (MA, USA). Antibodies against GAPDH (#MB001) were purchased from Bioworld (Missouri, USA).

Zhao Y., Fan S., Zhu H., Zhao Q., Fang Z., Xu D., Lin W., Lin L., Hu X., Wu G., Min J, & Liang G. (2024). Podocyte OTUD5 alleviates diabetic kidney disease through deubiquitinating TAK1 and reducing podocyte inflammation and injury. Nature communications, 15(1).

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Immunohistochemical Evaluation of Molecular Markers

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Immunohistochemical (IHC) staining was conducted using DAB substrate kit (ZSGB-BIO, Beijing, China) and the antibodies against Ki-67 (rabbit IgG, 1:150 dilution; Proteintech Group, Inc., Rosemont, IL, USA), PCNA, TTF-1 and Tg (Mouse monoclonal IgG, 1:50 dilution; Santa Cruz Biotech, Santa Cruz, CA, USA), NF-κB, COX-2 and IL-6 (Mouse monoclonal IgG, 1:50 dilution; Santa Cruz Biotech, Santa Cruz, CA, USA) and IkBα (1:100 from Cell Signaling, Cat., Danvers, MA, USA), respectively [26 (link)]. The results were evaluated according to the labeling intensity and scored as negative (−), weakly positive (+), moderately positive (++), and strongly positive (+++).

Zheng X., Jia B., Song X., Kong Q.Y., Wu M.L., Qiu Z.W., Li H, & Liu J. (2018). Preventive Potential of Resveratrol in Carcinogen-Induced Rat Thyroid Tumorigenesis. Nutrients, 10(3), 279.

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Exosome Characterization and Uptake

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The qNano platform (iZON Science, UK) was used to analyze the distribution and absolute size of the exosomes. Morphological examination of isolated exosomes was performed through transmission electron microscopy (TEM; HT 00, Hitachi, Japan). The expression of CD9, CD63, and TSG101 in exosomes was evaluated by Western blot analysis, and the following primary antibodies were used: CD9, CD63, and TSG101 (1:1000; rabbit IgG, Proteintech). A green uorescent dye (DIO; Life Technologies) was utilized to label the exosomes in accordance with the manufacturer's instructions. Ultracentrifugation at 110,000 g at 4°C for 70 min was performed to remove excess dye. HBMECs were incubated with DIOlabeled exosomes at a concentration of 50 µg/ml for 8 hours, followed by the use of 4,6-diamidino-2phenylindole (DAPI; Southern Biotech, Birmingham, AL, USA) to stain nuclei, which were then analyzed by uorescence microscopy.

Xin W., Qiang S., Jianing D., Jiaming L., Fangqi L., Bin C., Yuanyuan C., Guowang Z., Jianguang X, & Xiaofeng L. (2021). Human Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Attenuate Blood-Spinal Cord Barrier Disruption via the TIMP2/MMP Pathway After Acute Spinal Cord Injury. Molecular neurobiology, 58(12).

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Modulation of Macropinocytosis and AMPK Signaling

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Cells were cultured in basal medium containing 3.151 g/L glucose, 1 mM pyruvate and 2.5 mM glutamine (Glc⁺/Gln⁺/Pyr⁺ medium, Biological Industries, Cat 01-172-1A), glucose, pyruvate and glutamine-free medium (Glc^ˉ/Gln^ˉ/Pyr^ˉ medium, Biological Industries, Cat 01-057-1A), or added 3.151 g/L glucose to Glc^ˉ/Gln^ˉ/Pyr^ˉ medium (Glc⁺/Gln^ˉ/Pyr^ˉ medium). For macropinocytosis inhibition, cells were first incubated with 100 μM EIPA (5-(N-ethyl-N-isopropyl)-Amiloride), a Na⁺/H⁺-exchanger inhibitor used for macropinocytosis inhibition) (MedChemExpress, Cat HY-101840) in Glc^ˉ/Gln^ˉ/Pyr^ˉ medium for 1 h at 26°C (toad cells) or 37°C (HepG2 cells), respectively. For AMPK signaling inhibition, cells were treated with 0–10 μM compound C or 0–20 μM SBI-0206965 (MedChemExpress, Cat HY-13418 and Cat HY-16966, respectively) in Glc^ˉ/Gln^ˉ/Pyr^ˉ medium for 3 h at 26°C. To deplete endogenous βγ-CAT, toad cells were incubated with 100 μg/mL anti-βγ-CAT rabbit polyclonal antibodies or 100 μg/mL rabbit IgG (Proteintech, Cat B900610) as the isotype control in Glc^ˉ/Gln^ˉ/Pyr^ˉ medium at 26°C.

Liu L.Z., Liu L., Shi Z.H., Bian X.L., Si Z.R., Wang Q.Q., Xiang Y, & Zhang Y. (2023). Amphibian pore-forming protein βγ-CAT drives extracellular nutrient scavenging under cell nutrient deficiency. iScience, 26(5), 106598.

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Investigating YAP and TRIM11 Interactions

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Total cell lysis of CAL62 were precleared with rabbit IgG for 2 h and the immunoprecipitated with YAP (Proteintech,13584-1-AP) or TRIM11 (Proteintech, 10851-1-AP) antibody overnight as previously described [29] , while rabbit IgG (Santa Cruz) was used as the negative control. The bounded protein was analyzed by Anti-YAP or Anti-TRIM11 antibody.
Protein stability assays CAL62 and KHM-5M cells were seeded in 24-well plates and transfected with siTRIM11 or siControl. After 24 h, cells were treated with 100 μM cycloheximide (MCE) for indicated time points. Western blot was performed to detect YAP degradation.

Tang J., Tian Z., Liao X., Cui Q., & Wu G. (2020). TRIM11 promotes the proliferation, migration and chemoresistance of anaplastic thyroid cancer by stabilizing YAP.

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Western Blot Analysis of DNA Damage Markers

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Protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and probed with antibodies against ATM (2873S, Cell Signaling Technology, CST); p-ATM -S1981 (AP0008, ABclonal); METTL3 (15073-1-AP, Proteintech); YTHDF1 (17479-1-AP, Proteintech); YTHDF2 (24744-1-AP, Proteintech); YTHDF3 (25537-1-AP, Proteintech); FTO (TA809392, OriGene); m6A (202003, Synaptic Systems); BRCA1 (20649-1-AP, Proteintech); H2A.X (A11361, ABclonal); γH2A.X (AP0687, ABclonal ); GAPDH(60004-1-Ig, Proteintech); eIF3A (3411T, CST); Flag (F1804, Sigma-Aldrich); anti‐Rabbit IgG HRP‐linked antibody (7074; CST); Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (SA00001-1, Proteintech); α-Tubulin (66031-1-Ig, Proteintech); CHK1(bs-1681R, Bioss); CHK2 (252092, Zenbio); p-CHK2-S19(AP0862, ABclonal); Anti-HA tag monoclonal antibody (TA100012, OriGene); Mouse-IgG (BA1046, Boster); Rabbit IgG (B900610, Proteintech); p21(10355-1-AP, Proteintech); 53BP1 (BA2878, Boster); CDK1 (D160158, BBI life sciences); CDK2 (D199431, BBI life sciences); Cyclin B2 (21644-1-AP, Proteintech).

Zhang X., Liu P., Zheng X., Wang J., Peng Q., Li Z., Wei L., Liu C., Wu Y., Wen Y., Yan Q, & Ma J. (2021). N6-methyladenosine regulates ATM expression and downstream signaling. Journal of Cancer, 12(23), 7041-7051.

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