Rabbit anti-hypusine antibody (ABS1064, Merck Millipore, USA, 1:5000), mouse anti-β-actin antibody (A1978, Sigma-Aldrich; 1:2500) and mouse anti-actin antibody (017–2455, Fujifilm Wako Pure Chemical Corporation; 1:5000) were used for detection of hypusine protein expression and as a loading control. Total OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam, Cambridge, UK; 1:1000) was used for detection of mitochondrial OXPHOS-complex expression and as a loading control. Protein denatured at 95 °C for 5 min were separated via SDS-PAGE on the 4–20% Mini-PROTEAN® TGX™ Gels (Bio-Lad Laboratories, Inc., USA), then electrophoretically transferred to Immuno-Blot® poly vinylidene difluoride membrane (Bio-Lad Laboratories). Protein denaturation was not performed to detect total OXPHOS. Protein was detected using horseradish peroxidase-labelled secondary antibodies and the enhanced chemiluminescence system. Band intensity was quantified using ImageJ software (version 1.47 v, National Institutes of Health, USA).
Abs1064
Manufactured by Merck Group
Sourced in United States
The ABS1064 is a laboratory instrument produced by Merck Group. It is designed to perform high-precision measurements and analyses. The core function of the ABS1064 is to provide accurate and reliable data for scientific research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
A1978, by Merck Group (1 mentions)
Total oxphos rodent wb antibody cocktail, by Abcam (1 mentions)
Enhanced chemiluminescent western blotting detection reagent, by GE Healthcare (1 mentions)
Sc 8334, by Santa Cruz Biotechnology (1 mentions)
A5441, by Merck Group (1 mentions)
Bradford protein assay dye reagent, by Bio-Rad (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ta505100, by OriGene (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Pierce direct magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using abs1064
1
Protein Expression and Mitochondrial OXPHOS Analysis
2
Comprehensive Protein and Gene Expression Analysis
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All the protocols for protein extraction, 2D and 1D protein electrophoresis, immunoblot detection, and quantitative gene expression studies based on reverse transcriptase and quantitative PCR (RTqPCR) including housekeeping genes for normalization were described previously (Belda-Palazón et al., 2014 (link)). The primers used for qPCR not reported previously are indicated in Table 1 and were designed using the web tool pcrEfficiency (Mallona et al., 2011 (link)). Relative gene expression values and statistical analysis was performed using the REST 2009 software program (Pfaffl et al., 2002 (link)) available from Qiagen (http://www.qiagen.com ). The anti-hypusine antibody ABS1064 from Merck Millipore (USA) was used according to the manufacturer.
3
Western Blot Analysis of Cellular Proteins
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Cells were washed with ice-cold PBS, then scraped from the surface of the culturing dishes and collected by centrifugation. Total protein lysates were extracted by RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Then, the Bradford assay was used to estimate protein concentrations. Protein samples were loaded onto 10% SDS-PAGE gels and transferred to the PVDF membranes. Membranes were blocked with 5% nonfat milk in Tris-Buffered Saline containing 0.05% Tween 20 (TBST) for 1.5 h and then incubated overnight with the primary antibodies at 4 °C. Antibodies against GFP (1:1000, sc-8334, Santa Cruz, CA, USA), hypusine (1:1000, ABS1064, Millipore, Temecula, CA, USA), and β-actin (1:20000, A5441, Sigma-Aldrich) were used in our study. On the next day, after washing three times with 1×TBST, membranes were incubated for 1.5 h with secondary antibodies at room temperature. Protein signals were visualized by Enhanced Chemiluminescent Western Blotting Detection Reagents (GE Healthcare) under a Chemiluminescent Imaging System (Tanon 5200, Tanon Corporation, Minhang, Shanghai, China). The full membranes are shown in Figures S3 and S4 .
4
Western Blot Analysis of Hypusine
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Mouse brain tissue was prepared in IP Lysis buffer provided in the Pierce™ Direct Magnetic IP/Co-IP Kit (ThermoFisher Scientific). Total protein concentration was determined using the Bradford dye reagent protein assay (Bio-Rad Laboratories). Cell lysates in SDS sample buffer were boiled for 10 min and equal amounts of protein were resolved by 12% SDS-PAGE. Protein was electro-transferred onto 0.45 µM polyvinylidene difluoride Immobilon-P membrane (Millipore). Primary antibodies, rabbit anti-Hypusine (ABS1064, Millipore), mouse anti-eIF5A2 (TA505100, Origene) and mouse anti-β-actin (SC-47778, Santa Cruz Biotechnology) were incubated overnight at 4°C in 5% BSA in Tris-buffered saline containing 0.1% Tween-20. The anti-hypusine antibody identifies the hypusine modification as well as the deoxyhypusine intermediate. The mouse anti-eIF5A2 antibody (TA505100, Origene) was tested to be specific for eIF5A2 protein only and did not detect eIF5A1 protein by western blot when using commercially available HEK293 cell lysates that overexpress either eIF5A1 or eIF5A2 (LC419616 or LC412495, Origene) (Fig. S5 ). Secondary antibodies were incubated for 1 h at room temperature in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20. Blots were imaged using an Odyssey Clx (Licor) western blot scanner.
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