Cells were plated at a density of 5 × 106 cells/dish and treated with 20 μM SFN-Cys for 24 h. After washed with ice-cold PBS, cells were lysed on ice via Nondenaturing Lysis Buffer (C1050, Applygen, Beijing, China) with protease inhibitor cocktail (04693132001, Roche, Shanghai, China). After total proteins were quantified, equal amount of proteins were incubated with the fixed primary antibodies overnight at 4 °C. Then, the complexes were co-incubated with protein A/G agarose (sc-2003, SantaCruz, Dallas, Texas, USA) rotationally for 3 h at 4 °C and the proteins were isolated from the beads by centrifuging and boiled for 5 min. Western blot was used to recognize the conjugated proteins.
Protein a g agarose sc 2003
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Protein A/G agarose (sc-2003) is a laboratory product manufactured by Santa Cruz Biotechnology. It is an agarose-based resin designed for the purification of immunoglobulins and immune complexes.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Mg132, by Merck Group (2 mentions)
A4596, by Merck Group (1 mentions)
Ni nta bead, by Qiagen (1 mentions)
Phosphate buffered saline (pbs), by Biosesang (1 mentions)
Ab25949, by Abcam (1 mentions)
Immobilon transfer membrane, by Merck Group (1 mentions)
Sh30022, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Genview (1 mentions)
V900854, by Merck Group (1 mentions)
F4799, by Merck Group (1 mentions)
Lps e coli o111 b4, by Merck Group (1 mentions)
Anti flag f3165, by Merck Group (1 mentions)
Anti β actin 66009 1 ig, by Proteintech (1 mentions)
Anti ub sc 8017, by Santa Cruz Biotechnology (1 mentions)
Anti cd44 im7, by BioLegend (1 mentions)
Anti cd62l mel 14, by BioLegend (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
10 protocols using protein a g agarose sc 2003
1
Immunoprecipitation and Western Blot Analysis
2
Co-immunoprecipitation of NPM1 and PARP1
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For co-IP, the cells were first transfected with plasmids bearing Flag and/or Myc tagged protein genes. Cells were then lysed and the supernatants were incubated with anti-Flag-agarose beads (A4596; Sigma, St. Louis, MO, USA; 20 μL) or anti-Myc-agarose beads (ab1253, Abcam; 20 μL) overnight at 4 °C, and the precipitates were washed five times with RIPA. To investigate the interaction between endogenous NPM1 and PARP1, the supernatants of cell lysates were first incubated with an NPM1 antibody for 2 h at 4 °C. Protein A/G-agarose (sc-2003; Santa Cruz, CA, USA; 20 μL) was then added and incubated overnight. The precipitates were washed five times with RIPA and analyzed by Western blotting. For IP in purified proteins, purified Flag-NPM1 protein was mixed with purified Myc-PARP1 protein. Anti-Myc magnetic beads were added to the mixture and cultured at 4 °C overnight. The precipitates were then washed five times with RIPA and analyzed by Western blot.
3
Investigating hGH Ubiquitination in Blood
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hGH-His was constructed by removing the stop codon from the aforementioned pET-28a-hGH using site-directed mutagenesis for PCR. The protein was purified by the same purification method as described above. In addition, then, to investigate whether hGH is ubiquitinated in the blood stream, 300 μL mouse whole blood was cleared for 1 h at 4 °C using 300 μL protein A/G Agarose (sc-2003, Santa Cruz), then centrifuged twice at 3000 rpm for 1 min to obtain supernatants. An amount of 100 ng of purified hGH-His was incubated for 1 h at 37 °C with MG132 (Sigma-Aldrich) (5 μg/mL) in 1.3 mL mouse whole blood and pull-down assay was performed using 30 μL Ni-NTA beads (Qiagen, Hilden, Germany). Then, the precipitant for hGH-His was washed five times with phosphate-buffered saline (PBS, Biosesang, Seongnam-si, Korea).
4
Rbpjl ChIP-qPCR on Pancreatic Acinar Cells
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Cultured pancreatic acinar cells were cross-linked with 1% paraformaldehyde at room temperature for 5 min, and then added with glycine at a final concentration of 127 mM to terminate the cross-linking. Next, the cells were lysed with cell lysis buffer (50 mM Tris HCl, pH = 8.1, 1% sodium dodecyl sulfate, 10 mM EDTA) containing protease inhibitor on ice for 30 min, and then subjected to ultrasonification to produce 200—1000 bp chromatin fragments.
Afterwards, ChIP Assay Kits (#9002, CST) were utilized. Protein A/G agarose (SC-2003, Santa Cruz Biotechnology, Inc, Santa Cruz, CA) and ChIP antibody to Rbpjl (ab25949, Abcam) or IgG (#14,705, CST) were added to 50 μg DNA and incubated overnight at 4 °C. Normal IgG antibody was adopted as NC. The DNA complex was washed successively with low-salt buffer and high-salt buffer. DNA was subsequently extracted and purified with phenol/chloroform. Specific ChIP-RT-qPCR primers (forward: 5′-TTGGAAAGGATGGAACCGGC-3′; reverse: 5′-CTTTGCAGCTAGGGGCTGAT-3′) were designed for the Arid5a promoter.
Afterwards, ChIP Assay Kits (#9002, CST) were utilized. Protein A/G agarose (SC-2003, Santa Cruz Biotechnology, Inc, Santa Cruz, CA) and ChIP antibody to Rbpjl (ab25949, Abcam) or IgG (#14,705, CST) were added to 50 μg DNA and incubated overnight at 4 °C. Normal IgG antibody was adopted as NC. The DNA complex was washed successively with low-salt buffer and high-salt buffer. DNA was subsequently extracted and purified with phenol/chloroform. Specific ChIP-RT-qPCR primers (forward: 5′-TTGGAAAGGATGGAACCGGC-3′; reverse: 5′-CTTTGCAGCTAGGGGCTGAT-3′) were designed for the Arid5a promoter.
5
Immunoprecipitation and Western Blot Analysis
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For immunoprecipitations, cell extracts were prepared in Triton lysis buffer (20 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1% Triton X-100, and a protease inhibitor cocktail from Roche), and protein concentration was determined using a bicinchoninic acid assay (Pierce BCA protein assay kit, 23225). One-milligram aliquots of lysate protein was immunoprecipitated with 5 μg of antibodies overnight at 4°C. The immune complexes were captured with protein A/G-agarose (sc-2003; Santa Cruz Biotechnology) for an additional 2 h. Samples were prepared by boiling the beads in 2× sample buffer and loaded in SDS-PAGE gels, and the gels were transferred to Immobilon transfer membrane (Millipore; IPVHOOO10), which was blotted with corresponding antibodies and developed with enhanced chemiluminescence substrate (PerkinElmer; NEL05001EA).
6
Isolation and Culture of Porcine Vesicular Virus
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SVV-LNSY01-2017 used in this study was isolated from the pig with Porcine primary vesicular disease (PIVD). Baby hamster kidney cells (BHK21 cells; ATCC, CCL-10) were cultured in Dulbecco’s modified essential medium (HyClone, SH30022.01) containing 10% fetal bovine serum (Gibco, 16000044) and 100 U/ml of penicillin (GENVIEW, GA3502) at 37 °C in a humidified 5% CO2 incubator. Myeloma cells SP2/0 (ATCC, CRL-1581) were grown in RPMI 1640 medium (HyClone, SH30809.01) containing 10% FBS, 100 U/ml of penicillin grown under the same conditions as those described above. SPF level BalB/c mice were purchased from the Animal Experimental Center of Huazhong Agricultural University. Protein A/G Agarose (sc-2003) was purchased from Santa Cruz Biotechnology. Alexa Fluor 555 goat anti-mouse (A32727) antibodies were obtained from Invitrogen. Polyvinylidene fluoride (PVDF, 46978100) membranes were purchased from Roche.
7
Immunoprecipitation and Western Blotting
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Immunoprecipitation was performed as we previously described 26 (link),27 (link). Cell lysates were immunoprecipitated with TMEM16A, p62, LC3B-I/II, bcl-2, Beclin-1 and VPS3 antibodies (those described for the western blotting protocol) using protein A/G agarose (sc-2003; Santa Cruz Biotechnology). The immunoprecipitates were washed with lysis buffer thrice and subjected to western blotting analysis. The bound proteins were immunoblotted with the indicated antibodies.
8
Immunoprecipitation and Western Blotting
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HEK293T cells were transfected with the indicated plasmids, and then cells were washed with cold phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay buffer (V900854; Sigma, USA) containing Complete protease inhibitor cocktail (B14001; Biotool, USA) at 24 to 48 h posttransfection. The lysates were pretreated with 20 μl of protein A/G agarose (sc-2003; Santa Cruz, USA) for 1 h at 4°C, and then protein A/G agarose was removed by centrifugation. Two to three μg of the indicated antibody was added to the pretreated lysates, followed by overnight incubation at 4°C. Protein A/G agarose was added to the lysates and incubated at 4°C for another 2 h with rotation. The agarose beads were collected by centrifugation and washed four times with lysis buffer. The beads were resuspended in 1× SDS loading buffer and proteins were resolved by SDS-PAGE, followed by transferring to nitrocellulose and Western blotting.
9
Purification of Snail-Associated Proteins
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To purify Snail-associated proteins, pcDNA3.1-Flag-Snail plasmids were stably expressed in HEK-293T cells. A total of 5 × 109 cells were lysed in buffer A (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2.5 mM EDTA, 0.5%NP-40, 0.2 mM PMSF, and 0.5 mM dithiothreitol (DTT)). Cell lysates were precleared with the protein A/G agarose (sc-2003, Santa Cruz) for 2 h and then incubated with the anti-Flag M2 affinity gels (F2426, Sigma-Aldrich) at 0.5 ml of beads per 100 mg of cell lysate for 2 h to overnight with rotation. The anti-Flag M2 gels were washed four times with buffer BC500 containing 20 mM Tris-HCl (pH 7.8), 500 mM KCl, 0.2 mM EDTA, 10% glycerol, 10 mM β-mercaptoethanol, 0.2% NP-40, 0.2 mM PMSF, and protease inhibitor cocktail. The protein complexes were eluted with the 3×Flag peptides (F4799, Sigma-Aldrich) at 0.4 mg/ml in buffer BC100 containing 20 mM Tris-HCl (pH 7.8), 50 mM KCl, 0.2 mM EDTA, 10% glycerol, 10 mM β-mercaptoethanol, 0.2 mM PMSF, and protease inhibitor cocktail. The eluted proteins were resolved on 4 to 12% SDS-PAGE gels for Western blotting and colloidal staining analyses. The proteins were excised from the gels and identified by standard mass spectrometry.
10
Immune Signaling Pathway Activation
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LPS (E. coli O111:B4) and poly(I:C) were purchased from Sigma-Aldrich. Pam3CSK4 was purchased from InvivoGen. MG132 was obtained from Merck-Calbiochem. Escherichia coli O111:B4 was obtained from the China Center for Type Culture Collection. Anti-p-IκBα (9246), anti-p-p38 (9215), anti-p-JNK (9251), anti-p-ERK (4377), and anti-p-IRF3 (Ser 396, 29047) were purchased from Cell Signaling Technology. Anti-p38 (A11340), anti-JNK1 (A0288), anti-TLR4 (A5258), anti-MyD88 (A0980), anti-TRIF (A1155), and anti-ERK (A10613) antibodies were purchased from Abclonal Technology. Anti-S100A10 (11250-1-AP), anti-IRF3 (11312-1-AP), and anti-β-actin (66009-1-Ig) were purchased from Proteintech. Anti-Ub (sc-8017) and protein A/G agarose (sc-2003) used for immunoprecipitation were purchased from Santa Cruz Biotechnology. Anti-Flag (F3165) was purchased from Sigma. Anti-HA (901513, previously Covance catalog# MMS-101R) and anti-V5 (680601) were obtained from BioLegend. Fluorochrome-conjugated anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-B220 (RA3-6B2), anti-Ly-6G (1A8), anti-CD11b (M1/70), anti-CD11c (N418), anti-TLR4 (SA15-21), anti-F4/80 (BM8), anti-CD44 (IM7), and anti-CD62L (MEL-14) antibodies were purchased from BioLegend. Fluorochrome-conjugated anti-CD25 (PC61.5) and Foxp3 (FJK-16s) antibodies were purchased from eBioscience.
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