Mouse α gfp antibody

To prepare cell lysates, log-phase cells (OD₆₀₀ 2.0) were pelleted, resuspended in sample buffer (2% SDS, 1% 2-mercaptoethanol), boiled and lysed by glass-bead mechanical disruption [7 (link)]. The lysates were collected after centrifugation. After separating the proteins by 10% SDS–PAGE, samples were transferred to nitrocellulose or PVDF blocked with 5% skimmed milk in TBS-T (137 mM NaCl, 15.2 mM Tris-HCl, 4.54 mM Tris, 0.896 mM Tween 20) and then probed with primary and fluorescent secondary antibodies. Mouse α-GFP antibody was from Santa Cruz Biotechnology (1 : 2000, GFP(B-2):sc-9996). Peroxidase conjugated α-mouse IgG (1 : 5000; Sigma, A-4416) treated with ECL (Thermo Scientific) was used to develop the western blot.

For GFP-tagged KdmB, mouse α-GFP antibody (Cat# SC-9996, SantaCruz) was used at 1:1,000 dilution in blocking solution (TBST with 5% milk). Secondary goat α-mouse (Cat# 170–6516, Biorad) was used at 1:2,000 dilution in blocking solution. A rabbit polyclonal antibody against a subunit of the SCF complex, SkpA (SconC) was used (1:2,000 dilution in TBST with 5% milk) as a loading control with the Goat α-Rabbit (Bio-Rad, Cat# ab190479; 1:2,000) secondary antibody. For nuclear isolation, approximately 2x10⁶ spores were inoculated into GMM with required supplements for 24 h at 30 °C submerged culture. Buffers and extraction protocol were performed similarly as described previously (Soukup and Keller, 2013 ). Antibodies (1:2,000 dilution in blocking solution) used for histone PTMs were as following; α-H3 (Abcam; AB1791), α-H3K4me3 (Active Motif; 39159), α-H3K9me3 (Abcam; ab8898), α-H3K36me3 (Abcam; ab9050), α-H3K9ac (Merck; 07–352), α-H3K14ac (Merck; 07–353).