The anti-PARN antibody for western blot analysis was raised against purified, recombinant His-PARN and affinity purified with the antigen coupled to SulfoLink beads (Thermo Fisher Scientific). The anti-C21orf70 antibody was raised against a C-terminal peptide (C-GQTLARQMQLEDGGQL) and purified as described above. The antibody against RPL23a has been raised as previously described (40 (link)). The antibodies against DIM2, ENP1, NOB1, RPS3 (34 (link)), NOC4L, RRP12, RPS19 (29 (link)) and TSR1 (41 (link)) have been described previously. Anti-PARN for immunofluorescence was purchased from Abcam (ab89831), anti-β-actin and anti-α-tubulin from Sigma Aldrich (A1987 and T5168), anti-CK1δ and anti-HNRNPC from Abcam (AF12G4 and ab10294), anti-CK1ε (610445), anti-HA from Covance (MMS-101P) and anti-RPL10 from Santa Cruz Biotechnologies, Inc. (sc-798). Secondary antibodies for immunofluorescence were purchased from Invitrogen.
Sulfolink beads
Manufactured by Thermo Fisher Scientific
SulfoLink beads are a type of laboratory reagent used for protein immobilization and purification. They are based on crosslinked agarose beads that have been activated with sulfhydryl-reactive groups, allowing for the covalent attachment of sulfhydryl-containing biomolecules. SulfoLink beads provide a simple and effective method for the immobilization and purification of proteins, enzymes, and other sulfhydryl-containing molecules.
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4 protocols using sulfolink beads
1
Antibody Generation and Characterization for Western Blot and Immunofluorescence
2
Characterization of HeLa and HEK293 Cell Lines
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The HeLa RPS2-YFP, RPL29-GFP and HEK293 (FlpIn TRex, Invitrogen) HASt-GFP cell lines have been described previously (22 (link),24 (link),25 (link)). The HEK293 NOL10-StHA and NGDN-StHA cell lines for tandem affinity purification (TAP) were generated as previously described (25 (link)). HeLa K cells were obtained from D. Gerlich (IMBA, Vienna, Austria). HCT116 wt and p53−/− cell lines were kind gifts from B. Vogelstein (Johns Hopkins, Baltimore, USA) and have been described previously (26 (link)). All cell lines were grown in DMEM with 10% FCS and 1% penicillin/streptomycin at 37°C, 5% CO2.
α-AATF antibodies were raised against recombinant His-tagged full-length AATF in rabbits and purified using the antigen coupled to SulfoLink beads (Thermo Fisher Scientific). The following antibodies have been described previously: α-ENP1, α-NOB1, α-RPS3 (24 (link)); α-CRM1 (27 (link)); α-RPL23A (28 (link)). α-FBL (sc-166001) and α-NAT10 (sc-271770) were purchased from Santa Cruz Biotechnology; α-NOL10 (ab181161) from Abcam; α-NGDN (STJ24765) from St John's Laboratory; α-actin (A1978) from Sigma and α-HA (ENZ-ABS120) from Enzo Life Sciences. Leptomycin B (L-6100) was purchased from LC Laboratories.
α-AATF antibodies were raised against recombinant His-tagged full-length AATF in rabbits and purified using the antigen coupled to SulfoLink beads (Thermo Fisher Scientific). The following antibodies have been described previously: α-ENP1, α-NOB1, α-RPS3 (24 (link)); α-CRM1 (27 (link)); α-RPL23A (28 (link)). α-FBL (sc-166001) and α-NAT10 (sc-271770) were purchased from Santa Cruz Biotechnology; α-NOL10 (ab181161) from Abcam; α-NGDN (STJ24765) from St John's Laboratory; α-actin (A1978) from Sigma and α-HA (ENZ-ABS120) from Enzo Life Sciences. Leptomycin B (L-6100) was purchased from LC Laboratories.
3
Collagen Peptide Antibody Production
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19-amino acid peptides from the human collagen Iα1 C-terminal propeptide and C-terminal telopeptide were synthesized with an additional Cys residue at the N-terminus. This residue was used to link the peptides to KLH for immunization of rabbits and to Sulfolink beads (Thermo Fisher Scientific Inc., Rockford, IL). Total IgG was purified from rabbit serum. Specific IgG was then affinity purified from total IgG using the immunogenic peptides attached to Sulfolink beads. After elution, affinity-purified IgG was spin-concentrated and desalted into PBS.
4
Rabbit Antibody Production for Phosphopeptide
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Rabbits were injected bi‐weekly with AASpPAAGRC peptide conjugated to Imject Maleimide‐Activated Blue Carrier Protein (Thermo Fisher, 77664), and serum was collected after 4 months. Rabbit antibody was purified by first passing through phosphopeptide immobilized to Sulfolink beads (Thermo Fisher, 20401) and then subtracted using non‐phosphorylated peptide. Serum from 3 rabbits was collected and purified, and the serum with the highest antibody yield was used for subsequent experiments. Rabbit housing, injections and serum collection was performed by Pineda Antikörper‐Service (Berlin, Germany).
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