Brain or cortical lysates obtained from FVB-Tg(CAG-Diaph3)924/Lesp/J mice, which constitutively overexpress Diaph3, were prepared as described above. Proteins were quantified using the BCA kit (Pierce). Two hundred micrograms of proteins were incubated overnight in lysis buffer containing protease inhibitors and 5 μg rabbit anti-Diaph3 antibody. As negative control for the IP, the lysate was treated in the same conditions but without antibody. Immune complexes from anti-Diaph3 and control IP were recovered by incubation (2 h at 4 °C) with 20 μl protein A-coated agarose beads (Invitrogen) and centrifugation (2 min at 2,500 g). Beads were washed three times in PBS and proteins were eluted by addition of 5 × Laemmli buffer and heating at 95 °C for 5 min. The samples were processed for western blotting, for Survivin and BubR1 proteins. Total brain or cortical lysate were used to immunoprecipitate Survivin and BubR1, respectively.
Protein a coated agarose beads
Manufactured by Thermo Fisher Scientific
Protein A-coated agarose beads are a type of lab equipment used for the purification and isolation of immunoglobulins and other proteins that bind to Protein A. The beads are composed of agarose, a polysaccharide derived from seaweed, and are coated with Protein A, a bacterial protein that has a high affinity for the Fc region of immunoglobulins. These beads can be used in affinity chromatography and other protein purification techniques.
Automatically generated - may contain errors
3 protocols using protein a coated agarose beads
1
Immunoprecipitation of Diaph3 Interactors
2
ChIP-PCR for Histone Modifications
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We conducted ChIP as described in previous studies (Lämke et al, 2016a ). In short, roughly 500 mg of 10‐ and 12‐day‐old seedlings were cross‐linked by vacuum‐infiltrating 1% formaldehyde for 15 min. Formaldehyde was quenched using 2 M glycine. Samples were stored at −80°C until further processing. Further, nuclei extraction was performed and chromatin was sonicated using a Diagenode Bioruptor (medium setting, 14 cycles each with 30 s on/30 s off with ice cooling), yielding fragments with a size of around 250 bp. Antibodies (anti‐H3, ab1791; anti‐H3K4me3, from Abcam, ab8580 http://www.abcam.com ) were pre‐coupled to protein A‐coated agarose beads (Invitrogen) for at least 2 h at 4°C. Immunoprecipitations were done in IP buffer at 4°C for overnight. After, washing and reverse crosslinking, resulted DNA was extracted using the phenol–chloroform method and precipitated with ice chilled ethanol and glycogen (Invitrogen), and then re‐suspended in 20 µl of water. ChIP‐PCR was performed for three regions of APX2 and two regions of HSP18.2 gene loci. Amplification values were normalized to H3 (normalized signal modification/normalized signal H3). The given values in graphs are the means of three biological replicates, with each replicate was normalized to the respective 22°C control with no HS (NHS) sample before averaging.
3
ChIP-Seq protocol for histone modifications
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We conducted ChIP as described in previous studies (Lamke et al, 2016a) . In short, roughly 500 mg of 10 and 12 days old seedlings were cross-linked by vacuum-infiltrating 1% formaldehyde for 15 min. Formaldehyde was quenched using 2M glycine. Samples were stored at -80⁰C until further processing. Further, nuclei extraction was performed and chromatin was sonicated using a Diagenode Bioruptor (medium setting, 14 cycles each with 30 sec on/ 30 sec off with ice cooling), yielding fragments with a size of around 250 bp. Antibodies (anti-H3, ab1791; anti-H3K4me3, from Abcam, http://www.abcam.com) were pre-coupled to protein A-coated agarose beads (Invitrogen) for at least 2 h at 4⁰C. Immunoprecipitations were done in IP buffer at 4⁰C for overnight. After, washing and reverse crosslinking, resulted DNA was extracted using the phenol-chloroform method and precipitated with ice chilled ethanol and glycogen (Invitrogen), then re-suspended in 20 µl of water. ChIP-PCR was performed for three regions of APX2 and two regions of HSP18.2 gene loci. Amplification values were normalized to H3 (normalized signal modification/normalized signal H3). The given values in graphs are the means of three biological replicates, with each replicate was normalized to the respective 22⁰C control with no heat stress (NHS) sample before averaging.
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