Affi gel 10 gel

SDS PAGE and western blot analyses were performed essentially as previously described [19 (link)]. WB signal intensities were normalized to total proteins using densitometric analysis of Ponceau S stained membranes. Primary antibodies used were: α-HMGA1 (homemade) and α-H1.2 (ab181977, Abcam, Cambridge, UK). Secondary antibodies were: α-Rabbit IgG Peroxidase conjugate (#A0545, Sigma Aldrich, St. Louis, MO, USA) and α-Mouse IgG Peroxidase conjugate (#A9044, Sigma Aldrich, St. Louis, MO, USA). The α-HMGA1 antibody was produced by immunization of a rabbit with a recombinant HMGA1a protein lacking the C-terminal acidic domain. α-HMGA1 antibodies were affinity purified form serum using a resin (Affi-Gel 10 Gel #153-6046, Bio-Rad Laboratories, Hercules, CA, USA) covalently derivatized with HMGA1 recombinant protein. Recombinant proteins used for immunization and affinity purification were purified by reversed-phase HPLC and checked by mass spectrometry. Antibodies were eluted with a Glycine 0.2 M pH 2.8 solution and acidic pH was neutralized with a Tris 2 M pH 8 solution. Antibodies were stored at −20 °C in small aliquots.

For pull down assays mono-N-Boc protected 2,2'-(ethylenedioxy)bis(ethylamine) was attached to the carboxylic ester of efsevin and its derivatives through the amide bond. After removal of the Boc group using TFA, the primary amine was coupled to the carboxylic acid of Affi-Gel 10 Gel (Biorad, Hercules, CA). 2-day-old zebrafish embryos were deyolked by centrifugation before being lysed with Rubinfeld's lysis buffer (Rubinfeld et al., 1993 (link)). The lysate was precleaned by incubation with Affi-Gel 10 Gel to eliminate non-specific binding. Precleaned lysate was incubated with affinity beads overnight. Proteins were eluted from the affinity beads and separated on SDS-PAGE. Protein bands of interest were excised. Gel plugs were dehydrated in acetonitrile (ACN) and dried completely in a Speedvac. Samples were reduced and alkylated with 10 mM dithiotreitol and 10 mM TCEP solution in 50 mM NH₄HCO₃ (30 min at 56°C) and 100 mM iodoacetamide (45 min in dark), respectively. Gel plugs were washed with 50 mM NH₄HCO₃, dehydrated with ACN, and dried down in a Speedvac. Gel pieces were then swollen in digestion buffer containing 50 mM NH₄HCO₃, and 20.0 ng/μl of chymotrypsin (25°C, overnight). Peptides were extracted with 0.1% TFA in 50% ACN solution, dried down and resuspended in LC buffer A (0.1% formic acid, 2% ACN).