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Mouse anti puromycin

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Mouse anti-puromycin is a laboratory reagent used to detect the presence of puromycin, an antibiotic commonly used in cell culture experiments to select for cells that have undergone genetic modification. This antibody specifically binds to puromycin, allowing researchers to visualize and quantify its incorporation into newly synthesized proteins as a measure of protein translation activity.

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Lab products found in correlation

Mouse anti gapdh, by Merck Group (3 mentions) Goat anti kif17, by Santa Cruz Biotechnology (2 mentions) Itaq universal one step rt qpcr, by Bio-Rad (2 mentions) Cfx96, by Bio-Rad (2 mentions) Rabbit anti 4e bp1, by Cell Signaling Technology (2 mentions) Mouse anti g3bp1, by Santa Cruz Biotechnology (2 mentions) Rabbit anti eif2α, by Cell Signaling Technology (2 mentions) Rabbit anti phospho eif2α ser51, by Cell Signaling Technology (2 mentions) Mouse anti flag, by Merck Group (2 mentions) Revert 700 total protein stain, by LI COR (2 mentions) Rabbit anti caprin 1, by Proteintech (1 mentions) Goat anti eif3b, by Santa Cruz Biotechnology (1 mentions) Rabbit anti eif4g, by Cell Signaling Technology (1 mentions) Rat anti tubulin, by Abcam (1 mentions) Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions) Novex system, by Thermo Fisher Scientific (1 mentions) Mouse anti eif2α l57a5, by Cell Signaling Technology (1 mentions) Pvdf membrane, by Thermo Fisher Scientific (1 mentions) Odyssey fc system, by LI COR (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Puromycin (5590 citations) Anti-puromycin (84 citations) Mouse anti-puromycin monoclonal antibody (5 citations) Mouse anti-puromycin antibody (5 citations) Mouse monoclonal anti-puromycin (3 citations) Mouse anti-puromycin IgG 2a antibody (3 citations) Anti-puromycin mouse monoclonal antibodies (2 citations) Mouse anti-Puromycin (clone 12D10) primary antibody (2 citations) Monoclonal mouse anti-puromycin clone 12D10 (2 citations)

21 protocols using mouse anti puromycin

1

Antibody-Based Neuronal Protein Analysis

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Antibodies used were: rabbit anti-Sam68 (sc-333, SCBT, 1:500); mouse anti-Sam68 (sc-136062 mouse: SCBT); mouse anti-puromycin (Clone 12D10; Millipore; 1:20,000); mouse anti-PSD95 (clone K28/43, UC Davis, NeuroMab Facility 1:5000); chicken anti-MAP2 (EnCor Biotechnology, Cat# CPCA-MAP2); mouse anti-Arc (sc-17839, SCBT, 1:500); rabbit anti-Arc (156003, SYSY, 1:1000); mouse anti-RPS3 (sc-376098, SCBT, 1:1000); goat anti-KIF17 (Santa Cruz Biotechnology; 1:200) and mouse anti KIF5A (NKHC1, C-11, Santa Cruz Biotechnology; 1:200). RT-qPCR was performed on a CFX96 (BioRad) and iTaq Universal One-Step RT-qPCR (BioRad). Oligonucleotides used for RT-PCR were Arc (5′-ATGAATGGGCCAGCCAAGAA and 5′-TCCTCCTCAGCGTCCACATA), MAP2 (5′-CTGCCGGACCTGAAGAATGT and 5′-GCTTGGGGACTGTGTGATGA) and CaMKIIα (5′-GAAGATGTGCGACCCTGGAA and 5′-TGCGGATATAGGCGATGCAG). shRNAs used for knockdown of Sam68 (shS68) targeted the 3′UTR of Sam68; (5′-GTTATGAG CAAACTTGTTACT) and a non-targeting (shNT) sequence (5′-GCGTCACCAATGCGTTAATGG) as described (Klein et al., 2013 (link)). Adenoviral associated virus serotype 2 was generated in the Janelia Research Campus core facility using the shRNA sequences above.
Klein M.E., Younts T.J., Cobo C.F., Buxbaum A.R., Aow J., Erdjument-Bromage H., Richard S., Malinow R., Neubert T.A., Singer R.H., Castillo P.E, & Jordan B.A. (2019). Sam68 Enables Metabotropic Glutamate Receptor-Dependent LTD in Distal Dendritic Regions of CA1 Hippocampal Neurons. Cell reports, 29(7), 1789-1799.e6.
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2

Antibody-Based Neuronal Protein Analysis

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Antibodies used were: rabbit anti-Sam68 (sc-333, SCBT, 1:500); mouse anti-Sam68 (sc-136062 mouse: SCBT); mouse anti-puromycin (Clone 12D10; Millipore; 1:20,000); mouse anti-PSD95 (clone K28/43, UC Davis, NeuroMab Facility 1:5000); chicken anti-MAP2 (EnCor Biotechnology, Cat# CPCA-MAP2); mouse anti-Arc (sc-17839, SCBT, 1:500); rabbit anti-Arc (156003, SYSY, 1:1000); mouse anti-RPS3 (sc-376098, SCBT, 1:1000); goat anti-KIF17 (Santa Cruz Biotechnology; 1:200) and mouse anti KIF5A (NKHC1, C-11, Santa Cruz Biotechnology; 1:200). RT-qPCR was performed on a CFX96 (BioRad) and iTaq Universal One-Step RT-qPCR (BioRad). Oligonucleotides used for RT-PCR were Arc (5′-ATGAATGGGCCAGCCAAGAA and 5′-TCCTCCTCAGCGTCCACATA), MAP2 (5′-CTGCCGGACCTGAAGAATGT and 5′-GCTTGGGGACTGTGTGATGA) and CaMKIIα (5′-GAAGATGTGCGACCCTGGAA and 5′-TGCGGATATAGGCGATGCAG). shRNAs used for knockdown of Sam68 (shS68) targeted the 3′UTR of Sam68; (5′-GTTATGAG CAAACTTGTTACT) and a non-targeting (shNT) sequence (5′-GCGTCACCAATGCGTTAATGG) as described (Klein et al., 2013 (link)). Adenoviral associated virus serotype 2 was generated in the Janelia Research Campus core facility using the shRNA sequences above.
Klein M.E., Younts T.J., Cobo C.F., Buxbaum A.R., Aow J., Erdjument-Bromage H., Richard S., Malinow R., Neubert T.A., Singer R.H., Castillo P.E, & Jordan B.A. (2019). Sam68 Enables Metabotropic Glutamate Receptor-Dependent LTD in Distal Dendritic Regions of CA1 Hippocampal Neurons. Cell reports, 29(7), 1789-1799.e6.
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3

Molecular Profiling of Translation Regulation

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The following primary antibodies were used in this study: mouse anti-G3BP1 (Santa Cruz, sc-81940, Santa Cruz, CA, USA), goat anti-eIF4AI (Santa Cruz, sc-14211), mouse anti-eIF4E (P-2, Santa Cruz, sc-9976), rabbit anti-eIF4G (Cell Signaling #2498), goat anti-eIF3B (Santa Cruz, sc-16377), rabbit anti-Caprin1 (Proteintech, 15112-1-AP, Chicago, IL, USA), rabbit anti-phospho(S51)-eIF2α (Cell Signaling #9721), rabbit anti-eIF2α (Cell Signaling #9722, Danvers, MA, USA), rabbit anti-4E-BP1 (Cell Signaling, #53H11), rabbit anti-phospho-4E-BP1(Thr37/46) (Cell Signaling, #236B4), rabbit anti-AMPKalpha (Cell Signaling, #2603), rabbit anti-phospho-AMPKalpha (Cell Signaling, #2535), rat anti-tubulin (Abcam, #ab6160, Cambridge, UK), rabbit anti-Trx1 (FL-105, Santa Cruz sc-20146) (WB), rabbit anti-Trx1 (Cell Signaling #2429) (IF) and mouse anti-puromycin (Millipore MABE343).
Eiermann N., Stoecklin G, & Jovanovic B. (2022). Mitochondrial Inhibition by Sodium Azide Induces Assembly of eIF2α Phosphorylation-Independent Stress Granules in Mammalian Cells. International Journal of Molecular Sciences, 23(10), 5600.
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4

Western Blot Analysis of Cellular Proteins

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Cells were lysed for 30 min on ice in RIPA Lysis Buffer supplemented with phosphatase and protease inhibitor cocktails (Santa Cruz Biotechnology). Lysates were cleared by centrifugation at 17000g for 10 min at 4°C. Supernatants were removed and assayed for protein concentration using the Pierce BCA Protein Assay Kit (Thermo Fisher). Western blotting was performed using Novex system (Invitrogen). Equal amounts of proteins were subjected to SDS-PAGE and transferred to PVDF membranes (Thermo Fisher). Membranes were incubated with primary antibodies at 4°C overnight. Membranes were washed in TBS-Tween and then incubated with HRP conjugated anti-mouse or anti-rabbit secondary antibodies (Jackson Immunoresearch) for 1 hr at room temp and developed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher). Signal detection was captured using Odyssey Fc system (LI-COR; Figure S6). The following primary antibodies were used: mouse anti-eIF2B5 (B-7, 1:50; Santa Cruz Biotechnology); mouse anti-β-Actin (2D4H5, 1:3000 for WB; Proteintech); mouse anti-Puromycin (12D10, 1:1000; Millipore); mouse anti-EIF2α (L57A5, 1:500; Cell Signaling); rabbit anti-phospho-EIF2α Ser51 (119A11, 1:500; Cell Signaling); rabbit anti-FBXO32 (EPR9148(2), 1:500; Abcam); mouse anti-V5 (V5-10, 1:3000 for WB; Sigma-Aldrich).
Cai E.Y., Kufeld M.N., Schuster S., Arora S., Larkin M., Germanos A.A., Hsieh A.C, & Beronja S. (2020). Selective translation of cell fate regulators mediates tolerance to broad oncogenic stress. Cell stem cell, 27(2), 270-283.e7.
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5

Immunoblot Analysis of HIF, Glycolysis, and mTOR Signaling

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Rabbit anti-HIF-2α (Novus, Littleton, CO, USA), mouse anti-HIF-1α (BD, San Jose, CA, USA), rabbit anti-GLUT-1 (ThermoScientific, Villebon-sur-Yvette, France), mouse anti-cyclin D1, rabbit anti-p70S6K, rabbit anti-phospho-p70S6K (Thr389), rabbit anti-phospho-p70S6K (Thr421/Ser424), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-4E-BP1, rabbit anti-phospho 4E-BP1 (Ser65), rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Spns2 (Sigma), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-puromycin (Millipore, 12D10) were used as primary antibodies. Proteins were visualized by an ECL detection system (Perbio, Villebon-sur-Yvette, France) using anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG (Bio-Rad, Marnes-la-Coquette, France). Densitometry quantitation was determined using the Image J software (NIH, Bethesda, MD, USA).
Bouquerel P., Gstalder C., Müller D., Laurent J., Brizuela L., Sabbadini R.A., Malavaud B., Pyronnet S., Martineau Y., Ader I, & Cuvillier O. (2016). Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer. Oncogenesis, 5(3), e209-.
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6

Measuring Muscle Protein Synthesis using SUnSET

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In order to examine if MSTN treatment reduced muscle protein synthesis (MPS) rates compared to DM/CTL myotubes, the SUnSET method was employed [40 (link)]. Briefly, RIPA homogenates from 145 mm plates were subjected to 4-20% SDS-polyacrylamide gel electrophoresis using pre-casted gels (C.B.S. Scientific Company, San Diego, CA, USA). Proteins were transferred to polyvinylidene difluoride membranes (Whatman™, Westran® Clear Signal), and membranes were blocked for 1 h at room temperature with 5% nonfat milk powder. Mouse anti-puromycin (1:5,000; Millipore) was then incubated with membranes overnight at 4°C in 5% bovine serum albumin, and the following day membranes were incubated with anti-mouse IgG secondary antibodies (Cell Signaling, Danvers, MA, USA) at room temperature for 1 h. Membranes were then developed using an enhanced chemiluminescent reagent (Amersham, Pittsburgh, PA, USA), and band densitometry was performed through the use of a UVP Imager and associated densitometry software (UVP, LLC, Upland, CA, USA).
Mobley C.B., Fox C.D., Ferguson B.S., Amin R.H., Dalbo V.J., Baier S., Rathmacher J.A., Wilson J.M, & Roberts M.D. (2014). L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy. Journal of the International Society of Sports Nutrition, 11, 38.
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7

Antibody Panel for Cellular Analysis

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The following antibodies are used for WB, ICC or FC: 1:200 (WB) of rabbit anti-NG2 cyto (Stegmüller et al., 2002 (link)), 1:1,000 (WB) of mouse anti-Flag (Sigma, F1804); 1: 200 (FC) of anti-Flag-FITC (Sigma, F4049); 1:1,000 (WB) of rabbit anti-FMRP (Sigma, F4055); 1:1,000 (WB) of rabbit anti-eEEF2 antibody (Abcam, ab40812); 1:1,000 (WB) of rabbit anti-eIF4B (CST, 3592T); 1:1,000 (WB) of rabbit anti-phospho (Ser422) eIF4B (CST, 3591S); 1:200 (ICC) of rabbit anti-PCNA (NEB, 13110); 1:1,000 (WB) of mouse anti-Puromycin (Millipore, MABBE343); 1:5,000 (WB) of rabbit anti-GAPDH (Biomol, A-300-641A); 1:400 (WB) of mouse anti-Cyclin E (SCBT, E-4), 1:1,000 (WB) of rabbit anti-p70S6K1 (CST, 9202). For studying mTOR and p70S6K1 phosphorylation, substrate sampler kit (CST, 9862T) was used and the antibody dilutions were made for WB according to kit suggestions.
Nayak T., Trotter J, & Sakry D. (2018). The Intracellular Cleavage Product of the NG2 Proteoglycan Modulates Translation and Cell-Cycle Kinetics via Effects on mTORC1/FMRP Signaling. Frontiers in Cellular Neuroscience, 12, 231.
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8

Proinsulin Synthesis Measurement in Islets

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Total cellular protein was extracted from 60 islets or 106 cells per sample with RIPA buffer (1% Nonidet P40, 0.5% sodium doxycholate, 0.1% SDS, 1 × PBS, pH 8.0) containing 1×protease and phosphatase inhibitor cocktails (Sigma) and were boiled in 2×SDS sample buffer and then loaded onto 4–15% gels for Western blots. To measure newly synthesized proinsulin the SUnSET method was used [29] (link). Briefly, 10 µg/ml puromycin was added 15 minutes before harvest. Western blot was performed and proteins were probed with mouse anti-puromycin (1∶5000, Millipore) and rabbit anti-proinsulin C-peptide (1∶500, Bio Vision) primary antibodies, followed by IR800 anti-mouse and IR700 anti-rabbit secondary antibodies (1∶20000, LI-COR) for visualization of newly synthesized proteins co-localized with proinsulin using LI-COR Odyssey scanner. Other primary antibodies used in western blot analysis were: anti-PERK (1∶500, Cell Signaling), anti-ERp72 (1∶1000, Stressgen, Inc), anti-GRP78/BiP (1∶500, Santa Cruz, Inc.), anti-ERp57 (1∶500, Santa Cruz), anti-PDI (1∶500, Stressgen, Inc.).
Wang R., Munoz E.E., Zhu S., McGrath B.C, & Cavener D.R. (2014). Perk Gene Dosage Regulates Glucose Homeostasis by Modulating Pancreatic β-Cell Functions. PLoS ONE, 9(6), e99684.
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9

Immunostaining Protocol for Neuronal Markers

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For immunostaining neurons were washed twice with warm HBSS and then fixed with warm 4% PFA in HBSS for 10 min. Fixed cells were washed thrice with HBSS and permeabilized with 0.1% Triton X-100 in DPBS for 5 min and blocked for at least 30 min in blocking solution (2% FCS, 2% BSA, 0.2% fish gelatin (Sigma) in DPBS). The following primary antibodies were used overnight in 10 vol% blocking solution in DPBS: mouse anti-Ago2 (2E12-1C9) (1:500, WH0027161M1, Sigma), rabbit anti-Stau2 (1:500, selfmade (28 (link))), mouse anti-Stau2 (1:500, selfmade (28 (link))), mouse anti-Puromycin (1:500, 12D10, Millipore), mouse anti-Dcp1a (1:500, Sigma), rabbit anti-Ddx6 (1:500 (Rck), MLB), mouse anti-Pum2 (1:10 000, Abcam). The following secondary antibodies were used for 2 h in 10 vol% blocking solution in DPBS: donkey anti-mouse or rabbit AF488-, AF555- or AF647-conjugated antibodies (all Invitrogen). Coverslips were mounted on microscope slides with Fluoromount™ Aqueous Mounting Medium (Sigma).
Ehses J., Schlegel M., Schröger L., Schieweck R., Derdak S., Bilban M., Bauer K., Harner M, & Kiebler M.A. (2022). The dsRBP Staufen2 governs RNP assembly of neuronal Argonaute proteins. Nucleic Acids Research, 50(12), 7034-7047.
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10

Antibodies for ADAM15 Characterization

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Several antibodies directed against the extracellular part ADAM15 were used: a goat polyclonal (# AF935) and a mouse monoclonal (# mab945) from R&D Systems. Rabbit anti-ADAM15 (cytoplasmic domain, # ab84834), rabbit anti-PABP (# ab21060), rabbit anti-tubulin (# ab134185) and rabbit anti-calnexin antibody (# ab22595) from Abcam. Mouse anti-CD25 (# 174–820) from Ancell-Enzo Life Sciences GmbH. Mouse anti-puromycin (# MABE343, clone 12D10), mouse anti-myc antibody, (# 05–724), rabbit anti-alpha5 integrin (# AB1921) from Merck Millipore. Rabbit anti-FAK (# AHO0502) was from Invitrogen. Anti-Glutathion-S-Transferase peroxidase conjugate (# A7340) from Sigma-Aldrich.
Böhm B.B., Fehrl Y., Janczi T., Schneider N, & Burkhardt H. (2018). Cell adhesion-induced transient interaction of ADAM15 with poly(A) binding protein at the cell membrane colocalizes with mRNA translation. PLoS ONE, 13(9), e0203847.
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