Pierce protein a g agarose

Manufactured by Thermo Fisher Scientific

Sourced in United States

Pierce Protein A/G Agarose is a pre-packed agarose resin designed for the purification of antibodies and antibody-containing samples. The resin combines the binding properties of Protein A and Protein G, providing a versatile tool for a wide range of immunoaffinity applications.

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Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (6 mentions) Scientz iid ultrasonic homogenizer, by Ningbo Scientz Biotechnology (3 mentions) Protease inhibitor cocktail, by Roche (3 mentions) Dulbecco s pbs, by Merck Group (2 mentions) Histrap excel column, by Cytiva (2 mentions) Pfuse chig hg1, by InvivoGen (2 mentions) Pfuse2ss chig hk vectors, by InvivoGen (2 mentions) Superdex 200 increase 10 300, by Cytiva (2 mentions) Rabbit anti notch1 antibody, by Abcam (2 mentions) Chip assay kit, by Merck Group (2 mentions) Ripa minimum lysis buffer, by Merck Group (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions) Mouse il 15 elisa kit, by Neobioscience (1 mentions) Atp solution, by Thermo Fisher Scientific (1 mentions) Ovalbumin (ova), by InvivoGen (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Red blood cell lysis buffer, by Solarbio (1 mentions)

Spelling variants of this product

Agarose (403 citations) Protein G agarose (128 citations) Protein A/G agarose (127 citations) Protein A agarose (127 citations) Protein A/G (47 citations) Pierce Protein A/G agarose beads (25 citations) Pierce Protein G Agarose (22 citations) Pierce Protein A/G Plus Agarose (14 citations) Pierce Protein A agarose (11 citations) Pierce protein A/G magnetic agarose (3 citations)

42 protocols using pierce protein a g agarose

Recombinant IL-15 and IL-15Rα Protein Production

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The IL-15 and IL-15Rα encoded rAD was constructed by Shanghai Genechem Co., Ltd, China. Recombinant mouse IL-15 was provided by Abcam Inc., USA. Pierce^TM Protein A/G Agarose and ATP solution were obtained from ThermoFisher Scientific, USA. Red blood cell lysis buffer was supplied by Beijing Solarbio Science & Technology Co., Ltd, China. Mouse IL-15 ELISA kit was purchased from Neobioscience Technology Co, Ltd, China. Mouse IFN-γ ELISA kit was obtained from Shanghai Jianglai Industrial Limited by Share Ltd, China. OVA was supplied by InvivoGen, USA. Sulfo-Cy7 NHS ester was obtained from Lumiprobe Corporation, USA. LPS was bought from Sigma-Aldrich, USA. Cell culture flasks, plates, and glass bottom culture dishes were purchased from Wuxi NEST Biotechnology Co., Ltd, China. Scientz-IID Ultrasonic Homogenizer and Gene Electroporator Scientz-2C were provided by Ningbo Scientz Biotechnology Co., Ltd, China. All used reagents and solvents were of analytical standard grade unless stated otherwise.

Wang K., Zhang X., Ye H., Wang X., Fan Z., Lu Q., Li S., Zhao J., Zheng S., He Z., Ni Q., Chen X, & Sun J. (2023). Biomimetic nanovaccine-mediated multivalent IL-15 self-transpresentation (MIST) for potent and safe cancer immunotherapy. Nature Communications, 14, 6748.

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Protein Immunoprecipitation and Immunoblotting

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Cells were collected with trypsinization and lysed with lysis buffer (20 mM Tris-HCl pH 8.6, 100 mM NaCl, 20 mM KCl, 1.5 mM MgCl₂, 0.5% NP-40, cOmplete™ EDTA-free Protease Inhibitor Cocktail) on ice for 1 h and centrifuged at 16,000 × g for 30 min. After quantification using a BCA protein assay kit (ThermoFisher, #23225), 3 mg of total protein were rotated with antibodies at 4 °C overnight. Protein-antibody complexes were then captured with the Pierce^TM Protein A/G Agarose (ThermoFisher Scientific, #20421) at 4 °C for 2 h with rotation and beads were then rinsed with wash buffer (25 mM Tris, 150 mM NaCl, pH 7.2), boiled and subjected to immunoblotting analysis. Antibodies used in this study are shown in Supplementary Table 8.

Teng L., Feng Y.C., Guo S.T., Wang P.L., Qi T.F., Yue Y.M., Wang S.X., Zhang S.N., Tang C.X., La T., Zhang Y.Y., Zhao X.H., Gao J.N., Wei L.Y., Zhang D., Wang J.Y., Shi Y., Liu X.Y., Li J.M., Cao H., Liu T., Thorne R.F., Jin L., Shao F.M, & Zhang X.D. (2021). The pan-cancer lncRNA PLANE regulates an alternative splicing program to promote cancer pathogenesis. Nature Communications, 12, 3734.

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Rab14 and PKCι Interaction Characterization

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HEK cells were transfected with Rab14-GFP and Flag-PKCι. Cells were harvested and lysed in Pierce Lysis Buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, pH 7.4; Thermo Scientific, Rockford, IL). Lysates were cleared by incubating with control agarose resin for 1 h at 4°C and then incubated with 5 μg of rabbit anti-Rab14 antibody or rabbit IgG as control overnight at 4°C, followed by incubation with Pierce Protein A/G Agarose (Thermo Scientific). Input and immunoprecipitation fractions were analyzed using anti-PKCι antibody.

Lu R., Dalgalan D., Mandell E.K., Parker S.S., Ghosh S, & Wilson J.M. (2015). PKCι interacts with Rab14 and modulates epithelial barrier function through regulation of claudin-2 levels. Molecular Biology of the Cell, 26(8), 1523-1531.

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Methoprene-induced Hsp83-V5 Interactome

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UAS-Hsp83-V5 was co-transfected with the Act5c-Gal4 vector into cells using Effectene transfection reagents according to the manufacturer’s instructions. After 46 h, the old medium was replaced with fresh medium containing 10 μM methoprene, and the cells were incubated for an additional 2 h. The harvested cells were lysed in ice-cold NP-40 lysis buffer (supplemented with protease and phosphatase inhibitor cocktail and protease inhibitor cocktail) for immunoprecipitation. The lysates were incubated with V5 antibody for 4 h and then with Pierce protein A/G agarose (#UC277911, Thermo Fisher Scientific) overnight at 4°C. Beads were collected by slow centrifugation, washed 4 times with lysis buffer and resolved by 10% SDS-PAGE. The excised bands were used for protease digestion and mass spectrometry.

Gao Y., Chen N., Zhang X., Li E.Y., Luo W., Zhang J., Zhang W., Li S., Wang J, & Liu S. (2022). Juvenile Hormone Membrane Signaling Enhances its Intracellular Signaling Through Phosphorylation of Met and Hsp83. Frontiers in Physiology, 13, 872889.

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RNA Immunoprecipitation Assay

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The RIP assay was performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Cat#17–701) following instructions provided by the manufacturer. In brief, 2 × 10⁷ cells were lysed with equal pellet volume of RIP lysis buffer. 100 μl cell lysate was incubated with Pierce™ Protein A/G Agarose (ThermoFisher Scientific, Cat#20422) pre-coated with YBX1 antibodies at 4 °C overnight. Normal rabbit or mouse IgG was used as the negative control. For cells transfected with pEGFP-N1-YBX1(FL), pEGFP-N1-YBX1-ΔAP, pEGFP-N1-YBX1-ΔCSD or pEGFP-N1-YBX1-ΔCST plasmids, the lysates were incubated with anti-GFP mAb-Magnetic Agarose (MBL, Cat#D153-10; Nagoya, Japan). After being washed with RIP wash buffer, beads-bound immunocomplexes were prior to RNA isolation and immunoblotting analysis. Information of antibodies and primers used in this study is shown in Supplementary Table 1 and Supplementary Tables 3&4, respectively.

Wang Y., Feng Y.C., Gan Y., Teng L., Wang L., La T., Wang P., Gu Y., Yan L., Li N., Zhang L., Wang L., Thorne R.F., Zhang X.D., Cao H, & Shao F.M. (2022). LncRNA MILIP links YBX1 to translational activation of Snai1 and promotes metastasis in clear cell renal cell carcinoma. Journal of Experimental & Clinical Cancer Research : CR, 41, 260.

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Immunoprecipitation and RNA-seq of miRNA-binding proteins

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The RIP assay kit for miRNA (#RN1005, MBL) was used following the manufacturer’s protocol. HUVECs (80% confluence) from four 100 mm culture dishes were collected for each condition (17 (link), 18 (link)). Cell lysis was conducted in hypoxia or normoxia using a cell scraper and ice-cold PBS and mi-Lysis Buffer (+) was added to the cell pellet. Lysates were snap-frozen and stored at −70°C until use. For immunoprecipitation, 15 μg of anti-EIF2C2/AGO2 (#RN003M, MBL) or mouse IgG2a (isotype control; # M076-3, MBL) and agarose beads (Pierce Protein A/G Agarose, #20421, Thermo Scientific) were used. RNA was isolated using a miRNeasy Mini Kit (Qiagen, Hilden, Germany). 700 μl of QIAzol Lysis Reagent was added to the washed beads and the obtained RNA was eluted in 30 μl of nuclease-free water and used in the next generation RNA sequencing experiments. Prior to NGS, post-RIP and Input samples quality were verified with Western blot and qPCR as described in the protocol (Supplemental Figure 1).

Moszyńska A., Jaśkiewicz M., Serocki M., Cabaj A., Crossman D.K., Bartoszewska S., Gebert M., Dąbrowski M., Collawn J.F, & Bartoszewski R. (2022). The hypoxia induced changes in miRNA-mRNA in RNA-induced silencing complexes and HIF-2 induced miRNAs in human endothelial cells. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(7), e22412.

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Quantification of ADAR1 Protein Levels

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Various antibodies were used, including anti-ADAR1 rabbit polyclonal antibody (Thermo Fisher), anti-Normal rabbit IgG (Sigma Aldrich), anti-GAPDH rabbit monoclonal antibody (Abcam), anti-KYNU rabbit polyclonal antibody (Thermo Fisher), anti-ADAR1 mouse monoclonal antibody (Thermo Fisher). Secondary antibodies were used, including goat anti-mouse (Abcam) and goat anti-rabbit IgG HRP (Thermo Fisher). For western blotting analysis, the primary antibodies dilutions were 1:1000 and GAPDH was used as the loading control. Secondary antibodies for western blotting analysis were diluted to 1:7000. Additional reagents used include pierce protein A/G agarose (Thermo Fisher), Protease inhibitor cocktail, EDTA-Free (100X) (Thermo Fisher), RIPA buffer, Pierce ip lysis buffer (Thermo Fisher), Sterile conical centrifuge tubes 15 ml (Thermo Fisher), cell culture flask 25 cm (Thermo Fisher).

Binothman N., Aljadani M., Alghanem B., Refai M.Y., Rashid M., Al Tuwaijri A., Alsubhi N.H., Alrefaei G.I., Khan M.Y., Sonbul S.N., Aljoud F., Alhayyani S., Abdulal R.H., Ganash M, & Hashem A.M. (2023). Identification of novel interacts partners of ADAR1 enzyme mediating the oncogenic process in aggressive breast cancer. Scientific Reports, 13, 8341.

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Coimmunoprecipitation of c-Cbl, p-Src, Caveolin-1, and LC3B

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Cells were lysed on ice in immunoprecipitation cell lysis buffer (Beyotime, P0031), containing a protease inhibitor cocktail (Roche, 049693132001). The lysate was centrifuged at 15,200 × g for 15 min at 4°C, and the supernatant was quantified by BCA (Thermo Fisher Scientific, 23235) analyses. For the coimmunoprecipitation assay, an amount of 500 μg of protein in 250 μL supernatant was incubated overnight at 4°C with anti-c-Cbl (1 : 50), anti-p-Src (1 : 50), anti-LC3B (1 : 50), and anti-caveolin-1 (1 : 50), followed by precipitation with 20 μL of Pierce® Protein A/G Agarose (Thermo Fisher Scientific, 20421) for 2 h at room temperature. Normal rabbit IgG was used as a negative control. The precipitated complexes were separated by SDS-PAGE gel and immunoblotted with anti-c-Cbl, anti-p-Src, anti-caveolin-1, and anti-LC3B. To reduce the signals from the denatured IP antibody, the secondary antibody anti-rabbit IgG light chain specific (Cell Signaling Technology, 93702) was used at 1 : 1000 dilution.

Bai X., Jia X., Lu Y., Zhu L., Zhao Y., Cheng W., Shu M, & Jin S. (2020). Salidroside-Mediated Autophagic Targeting of Active Src and Caveolin-1 Suppresses Low-Density Lipoprotein Transcytosis across Endothelial Cells. Oxidative Medicine and Cellular Longevity, 2020, 9595036.

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Immunoprecipitation of FLAG-tagged Proteins

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Cells were incubated with 2 mM DSP (dithiobis(succinimidyl propionate), Thermo Fisher Scientific, Waltham, MA, USA) as a crosslinker for 30 min. The crosslinker reaction was stopped with 20 mM Tris pH 7.5 for 15 min. Cells were lysed with RIPA buffer and protease-phosphatase inhibitors. Proteins were collected by centrifugation for 15 min at 19,000× g at 4 °C and pre-incubated with Pierce™ Protein A/G Agarose (Thermo Fisher Scientific) beads for 1 h at 4 °C on a rotator and incubated with anti-FLAG^® or normal IgG antibodies for 2 h at 4 °C on a rotator. Antibody-bound proteins were incubated with beads overnight at 4 °C on a rotator. Beads were recovered by centrifugation at 1100× g and washed with cold RIPA buffer. Immunoprecipitated proteins were resolved by immunoblot using Clean-Blot™ IP Detection Kit HRP (Thermo Fisher Scientific). Antibodies are described in Appendix C.

Jiménez-Martínez M., Ostalé C.M., van der Burg L.R., Galán-Martínez J., Hardwick J.C., López-Pérez R., Hawinkels L.J., Stamatakis K, & Fresno M. (2019). DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression. Cancers, 11(11), 1767.

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Recombinant Antibody Production and Purification

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cDNAs encoding the BCR heavy and light variable regions were synthesized by GeneScript and subcloned into pFUSE-CHIg-HG1 and pFUSE2ss-CHIg-hK vectors (InvivoGen), respectively. Full-length antibodies were expressed using ExpiCHO cells according to standard protocol. Cells were transfected with 0.6 μg/mL light chain plasmid and 0.4 μg/mL heavy chain plasmid. The medium was collected at Day 8 post-transfection. Antibodies from the medium were purified using Pierce Protein A/G Agarose (ThermoFisher, Cat# 20424) with the standard protocol. Filtered medium (0.22 μm) was loaded onto Protein A/G Agarose pre-equilibrated with binding buffer (25 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA). After washing with the same buffer for three times, the protein was eluted with 5 CV of 0.1 M Glycine pH 2.7 and the pH of eluent was immediately adjusted with 0.5 CV 1 M Tris pH 8.0. Protein was concentrated and further applied to Superdex 200 10/300 Increase (Cytiva) in Dulbecco’s PBS (Sigma) buffer. For SW186 Fab protein, the cDNA of heavy chain and light chain was subcloned into modified pCAG-His vector with an IL2 signal peptide at N-terminus. A stop codon was introduced to the C-terminus of light chain to make it a tag-free protein. The protein was then expressed the same way as full-length antibodies, and purified with HisTrap Excel column (Cytiva) using the AKTA system.

Fang Y., Sun P., Xie X., Du M., Du F., Ye J., Kalveram B.K., Plante J.A., Plante K.S., Li B., Bai X.C., Shi P.Y, & Chen Z.J. (2022). An antibody that neutralizes SARS-CoV-1 and SARS-CoV-2 by binding to a conserved spike epitope outside the receptor binding motif. Science Immunology.

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