Total protein concentration from transfected HEK-293 cells lysates was determined using the Pierce™ BCA protein assay kit (Thermo Fisher scientific). Protein lysates (100 µg) were mixed with 40 µl of protein A agarose slurry (Santa Cruz Biotechnology, sc-2001) and 2 µl of anti-PP2A C subunit antibody (Sigma-Aldrich, 05-421) in a total of 500 µl with pNPP Ser/Thr assay buffer (Sigma-Aldrich, 20-179), and incubated for 2 h at 4°C with constant rotation. Next, beads were washed three times with TBS and once with pNPP Ser/Thr assay buffer. Subsequently, threonine phosphopeptide (Sigma-Aldrich, 12-219) was added to the washed beads and pNPP Ser/Thr assay buffer to a final concentration of 500 µM as substrate for the enzymatic reaction, and it was incubated for 10 min at 30°C in a shaking incubator. Twenty-five microlitres of the enzymatic reaction were mixed with 100 µl of Malachite Green Phosphate Detection Solution (solution A and additive, Sigma-Aldrich, 20–105 and 20–104, respectively) in a Corning® 96-well half-area microplate (Merck, CLS3695-25EA), and incubated for 10 min at room temperature. Absorbance was measured at a wavelength of 650 nm in a Varioskan microtitre plate reader (ThermoFisher) and compared with the Phosphate standard (Sigma-Aldrich, 20-103).
Protein a agarose slurry
Manufactured by Santa Cruz Biotechnology
Protein A-agarose slurry is a laboratory product used for the purification of antibodies. It consists of Protein A, a bacterial-derived protein, covalently linked to an agarose bead matrix. The slurry form allows for easy handling and application in various antibody purification protocols.
Automatically generated - may contain errors
3 protocols using protein a agarose slurry
1
Quantifying PP2A Activity in HEK-293 Cells
2
Immunoprecipitation of Keratins 8/18
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Cell pellets were lysed in RIPA buffer. In 1 ml total volume, 1 mg of protein lysate was incubated with 6 μg of anti-K8/K18 antibody, purchased from BioLegend (1E8) for 4 h at 4 °C. 60 μl of protein A-agarose slurry (Santa Cruz) was then added and further incubated overnight at 4 °C. The beads were separated from the lysate using centrifugation and were washed 5 times using lysis buffer. After the last wash beads were treated with 1 X LDS sample buffer and heated at 90 °C for 10 minutes. IP samples were analyzed by SDS-PAGE and immunoblot analysis as previously described.
3
Immunoprecipitation of SnRK2.4/SnRK2.10
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Immunoprecipitation was performed as described previously [77 (link)], with minor modifications. For every mg of protein from crude extract 50 μL of protein A-agarose slurry (Santa Cruz Biotechnology, www.scbt.com ) and 100 μg of antibodies was used. For analysis of SnRK2.4/SnRK2.10 activity immunoprecipitation was performed from 500 μg of crude protein extract.
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