293T cells were cultured in 10-cm dishes. When cell confluence reached 70~80%, cells were transiently transfected with GFP-nucleolin and its GFP vector control. Twenty four hours after the transfection, the cells were extracted by using polysomelysis buffer (10 mM HEPES pH 7; 100 mM KCl; 5 mM MgCl2; 25 mM EDTA; 0.5% IGEPAL; 2 mM DTT; 50 units/ml RNase OUT; 50 units/ml Superase IN; 0.2 mg/ml heparin; and complete proteinase inhibitor). The cell lysates were centrifuged at 14,000× g for 10 min at 4°C. The anti-GFP agarose beads A/G (Purchased from Vector laboratories, Burlingame, CA, USA ) were added into the supernatant and rotated overnight at 4°C in NET2 buffer (50 mMTris–HCl, pH 7.4, 150 mM sodium chloride, 1 mM magnesium chloride, 0.05% IGEPAL, 50 U/mL RNase OUT, 50 U/mL Superase IN, 1 mM dithiothreitol, and 30 mM EDTA). The beads were washed three times, and resuspended in 100 μL NET2 and 100 μL SDS-TE (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 2% sodium dodecyl sulfate) and then incubated at 55°C for 30 min, mixing occasionally. The RNAs in the buffer of the beads were extracted by phenol-chloroform-isoamyl alcohol and RT-PCR was performed to identify the mRNA presented in the immune-complex.
Anti gfp agarose beads a g
Manufactured by Vector Laboratories
Sourced in United States
Anti-GFP agarose beads A/G are a solid-phase affinity matrix designed for the purification and detection of Green Fluorescent Protein (GFP) and GFP-fusion proteins. The beads consist of agarose beads coupled with a recombinant protein A/G, which can bind to the Fc region of antibodies. This allows for the efficient capture and isolation of GFP-tagged proteins from various sample sources.
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Lab products found in correlation
4 protocols using anti gfp agarose beads a g
1
Affinity Purification of Nucleolin-Bound mRNAs
2
GFP-NCL Immunoprecipitation and RT-PCR
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293 T cells were cultured in 10 cm dishes. When cell confluence reached 70–80%, cells were transiently transfected with GFP-NCL and its vector control. Twenty-four hours after the transfection, the cells were extracted with polysomelysis buffer (10 mM HEPES pH 7; 100 mM KCl; 5 mM MgCl2; 25 mM EDTA; 0.5% IGEPAL; 2 mM DTT; 50 units/ml RNase OUT; 50 units/ml Superase IN; 0.2 mg/ml heparin; and complete proteinase inhibitor). The cell lysates were centrifuged at 14,000 × g for 10 min at 4 °C. The anti-GFP agarose beads A/G (Vector laboratories, Burlingame, CA, USA) were added into the supernatant and rotated overnight at 4 °C in NET2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 1 mM magnesium chloride, 0.05% IGEPAL, 50 U/mL RNase OUT, 50 U/mL Superase IN, 1 mM dithiothreitol, and 30 mM EDTA). The beads were washed three times, and resuspended in 100 μL NET2 and 100 μL SDS-TE (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 2% sodium dodecyl sulfate) and then incubated at 55 °C for 30 min, mixing occasionally. The RNAs in the buffer of the beads were extracted by phenol-chloroform- isoamyl alcohol and RT-PCR was performed to identify the mRNA presented in the immune-complex.
3
RNA-Immunoprecipitation Assay in 293T Cells
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RNA-IP assay was performed as described previously [64 (link)]. The transient transfection efficiency of 293T cells is very high and convenient for transient transfection, so we use 293T to carry out RNA-IP assay. In brief, twenty-four hours after the transfection, 293T cells were extracted using polysome lysis buffer. Anti-GFP agarose beads A/G (Vector Laboratories, Burlingame, CA, USA) were added to the supernatant and rotated overnight at 4 °C. The beads were washed three times and re-suspended, then incubated at 55 °C for 30 min, occasionally mixed. RNA was extracted by TRIzol, and the mRNA present in the immune complex was identified by RT-PCR.
4
GFP-NCL RNA Immunoprecipitation Assay
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RNA-IP assay was performed as described previously [79 (link)]. Briefly, 293T cells were cultured in 10-cm dishes. When cell confluence reached 70∼80%, cells were transiently transfected with GFP-NCL and its GFP vector control. Twenty four hours after the transfection, the cells were extracted by using polysomelysis buffer (10 mM HEPES pH 7; 100 mM KCl; 5 mM MgCl2; 25 mM EDTA; 0.5% IGEPAL; 2 mM DTT; 50 units/ml RNase OUT; 50 units/ml Superase IN; 0.2 mg/ml heparin; and complete proteinase inhibitor). The cell lysates were centrifuged at 14,000 × g for 10 min at 4°C. The anti-GFP agarose beads A/G (Vector laboratories, Burlingame, CA, USA) were added into the supernatant and rotated overnight at 4°C in NET2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 1 mM magnesium chloride, 0.05% IGEPAL, 50 U/mL RNase OUT, 50 U/mL Superase IN, 1 mM dithiothreitol, and 30 mM EDTA). The beads were washed three times, and resuspended in 100 μL NET2 and 100 μL SDS-TE (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 2% sodium dodecyl sulfate) and then incubated at 55°C for 30 min, mixing occasionally. The RNAs in the buffer of the beads were extracted by phenol-chloroform-isoamyl alcohol and RT-PCR was performed to identify the mRNA presented in the immune-complex.
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