Antibody Generation and Validation for Epigenetic Regulators

Polyclonal antibodies for SAMD1 and L3MBTL3 were made using purified glutathione S-transferase (GST)–fusion proteins as antigen. For SAMD1, the antibody is directed against the SAM domain of human SAMD1 (amino acids 452 to 538). For L3MBTL3, the antibodies are directed against the N terminus (amino acids 3 to 233) or the C terminus (amino acids 778 to 883) of mouse L3MBTL3. Antibodies were made with Eurogentec using the 28-day speedy protocol. Obtained antibodies were affinity-purified. The following commercial antibodies were used: H3K4me3 (Diagenode, C15410003), H3K4me2 (Diagenode, C15410035), H3K27ac (Active Motif, 39133), H3K27me3 (Diagenode, C15410195), KDM1A (Abcam, 17721), tubulin (Millipore, MAB3408), Pou5f1/Oct4 (Santa Cruz Biotechnology, SC-5279), FLAG (Sigma-Aldrich, F3165), hemagglutinin (Roche, 11867423001), H2AUb1 (Cell Signaling Technology, 8240), Lamin B (Santa Cruz Biotechnology, sc-6217), EZH2 (Diagenode, C15410039), L3MBTL2 (Active Motif, 39569), PCGF6 (ProteinTech, 24103-1-AP), RING2 (Abcam, ab101273), RYBP (Sigma-Aldrich, PRS2227), SFMBT1 (Bethyl Laboratories, A303-221A), rabbit immunoglobulin G (IgG) control (Diagenode, C15410206), and H2Av (Drosophila) (Active Motif, 39716). The Sp1 antibody was described previously (42 (link)).

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Stielow B., Zhou Y., Cao Y., Simon C., Pogoda H.M., Jiang J., Ren Y., Phanor S.K., Rohner I., Nist A., Stiewe T., Hammerschmidt M., Shi Y., Bulyk M.L., Wang Z, & Liefke R. (2021). The SAM domain-containing protein 1 (SAMD1) acts as a repressive chromatin regulator at unmethylated CpG islands. Science Advances, 7(20), eabf2229.

Publication 2021

H3K4me3 H3K4me2 H3K27ac H3K27me3 KDM1A tubulin FLAG hemagglutinin Lamin B L3MBTL2

Amino acids Antibodies Antibody Antigen Drosophila Ezh2 Glutathione s transferase H3k4me3 Hemagglutinin Human Immunoglobulin g Kdm1a Lamin b Mouse Oct4 Proteins Rabbit Rybp Sam domain Tubulin

Corresponding Organization : Universitätsklinikum Gießen und Marburg

Other organizations : University of Cologne, Brigham and Women's Hospital, Harvard University, German Center for Lung Research, Boston Children's Hospital, University of Oxford, Ludwig Cancer Research

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Variable analysis

independent variables

Purified glutathione S-transferase (GST)–fusion proteins as antigen for making polyclonal antibodies against SAMD1 and L3MBTL3

dependent variables

Antibodies directed against the SAM domain of human SAMD1 (amino acids 452 to 538)
Antibodies directed against the N terminus (amino acids 3 to 233) or the C terminus (amino acids 778 to 883) of mouse L3MBTL3

control variables

Rabbit immunoglobulin G (IgG) control
H2Av (Drosophila) as a control

positive controls

Commercially available antibodies: H3K4me3, H3K4me2, H3K27ac, H3K27me3, KDM1A, tubulin, Pou5f1/Oct4, FLAG, hemagglutinin, H2AUb1, Lamin B, EZH2, L3MBTL2, PCGF6, RING2, RYBP, SFMBT1, Sp1

negative controls

Rabbit immunoglobulin G (IgG) control

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