Cell lysates (obtained after transfection of the pCMV-3AG-3a-EV or pCMV-3AG-3a-CD200 construct) containing 1 mg of protein were precleared by incubation with 40 μL of protein-A/G linked agarose beads (Santa Cruz) for 1 h at 4°C. After the beads were pelleted by centrifugation, the supernatant was incubated with 40 μL of anti-FLAG M2 affinity gel (Sigma-Aldrich) overnight at 4°C. After incubation, the beads were washed 3 times in RIPA buffer before being dissolved in SDS-PAGE loading buffer. Then, western blot analysis was performed. For fractionation of cellular extracts, MEER cells were transfected with the pCMV vector or pCMV-mouseCD200 vector. Nuclear and cytoplasmic extracts were prepared as described previously.31 (link)
Protein a g linked agarose beads
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Protein-A/G linked agarose beads are a type of chromatography resin designed for the purification of antibodies. The beads are composed of agarose, a polysaccharide derived from seaweed, which has been covalently coupled with either Protein A or Protein G. These proteins bind to the Fc region of antibodies, allowing them to be captured and separated from other molecules in a sample.
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4 protocols using protein a g linked agarose beads
1
Immunoprecipitation and Cellular Fractionation
2
Immunoprecipitation of Galectin-3 and HSF-1
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Cell lysates containing 750 μg proteins was pre-cleared by incubation with 40 μl protein-A/G linked agarose beads (Santa Cruz, CA, USA) for 1 hour at 4°C. After the beads were spun down, the supernatant was incubated with 1 μg its specific antibody (anti-galectin-3 and anti-HSF-1) overnight at 4°C, followed by incubation with 40 μl protein-A/G agarose beads for 1 hour. Mouse IgG (Santa Cruz, CA, USA) was used as the negative control. After the incubation, beads were washed three times in a RIPA buffer before being dissolved in a SDS-PAGE loading buffer. Western blot analysis was performed as described above.
3
Immunoprecipitation and Western Blot Analysis
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Cell lysate containing 750 μg of protein was pre-cleared by incubation with 40 μL of protein-A/G linked agarose beads (Santa Cruz) for 1 h at 4°C. After the beads were spun down, the supernatant was incubated with 1 μg of a specific antibody (anti-HA or anti-FLAG, respectively) overnight at 4°C, followed by incubation with 40 μL of protein-A/G agarose beads for 1 h. Mouse IgG (Santa Cruz) was used as the negative control. After the incubation, beads were washed 3 times in RIPA buffer before being dissolved in SDS-PAGE loading buffer. Western blot analysis was performed as described above.
4
Immunoprecipitation and Western Blot Analysis
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Cell lysates were pre-cleared by being incubated with 20 μl of protein A/G-linked agarose beads (Santa Cruz) for 1 hr at 4°C. After spinning down the beads, the supernatant was incubated with 2 μg of a specific antibody (anti-FLAG, anti-CD44, anti-Nanog, anti-Sox2, and anti-Oct4) overnight at 4°C, followed by incubation with 40 μl of protein A/G-linked agarose beads for 1 hr. A mouse or rabbit IgG (Santa Cruz) was used as the negative control. Following the incubation, the beads were washed three times in a RIPA buffer before being dissolved in a SDS-PAGE loading buffer. A western blot analysis was performed as described elsewhere [40 (link)].
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