After SDS–PAGE, proteins were transferred onto nitrocellulose (BioTrace NT; Pall Corp.) using the overnight wet transfer system (Amersham Biosciences), and Western blot analysis was performed as described previously (Guerriero et al., 2013 (link)) with the following primary antibodies: rabbit anti–glucose-6-phosphate dehydrogenase (G6PD) (A9521; Sigma-Aldrich) at 1:5000, polyclonal rabbit anti-Sec61p (Stirling et al., 1992 (link)) at 1:5000, rat anti–HA-peroxidase (Roche) at 1:10,000, monoclonal mouse anti-GFP antibody (Roche) at 1:5000, and mouse anti-Pma1, which was a gift from Amy Chang (University of Michigan, Ann Arbor, MI) at 1:1000. Secondary antibodies used in this study were as follows: HRP-conjugated goat anti-mouse or anti-rabbit secondary antibody (Cell Signaling Technology) at 1:5000. Proteins were visualized using SuperSignal Chemiluminescence (Thermo Scientific). Images of the blots were taken on a BIO-RAD ChemiDocXRS Image Station and quantified using ImageJ version 1.48v software (National Institutes of Health). Where indicated, protein half-life was calculated using PRISM GraphPad version 7.0C (GraphPad Software).
Rat anti ha peroxidase
Manufactured by Roche
Sourced in Germany
The Rat anti-HA-Peroxidase is a laboratory reagent used for the detection of proteins tagged with the HA (Hemagglutinin) epitope. It contains antibodies raised in rats that are specific to the HA tag, and are conjugated with the enzyme Peroxidase. This product can be used in various immunodetection techniques, such as Western blotting and immunohistochemistry, to visualize HA-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti tubulin, by Merck Group (3 mentions)
Mouse anti actin, by Abcam (3 mentions)
Mouse anti flag, by Merck Group (3 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Secondary hrp antibodies, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti gfp antibody, by Roche (1 mentions)
Biotrace nt, by Pall Corporation (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Roscovitine, by Selleck Chemicals (1 mentions)
Palbociclib, by Selleck Chemicals (1 mentions)
Mouse igg agarose, by Merck Group (1 mentions)
Rabbit anti flag, by Merck Group (1 mentions)
Sds sample buffer, by Thermo Fisher Scientific (1 mentions)
Mouse anti ty1, by Thermo Fisher Scientific (1 mentions)
9 protocols using rat anti ha peroxidase
1
Western Blot Analysis of Protein Expression
2
Ngn3 Protein Stability Kinetics
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Mouse insulinoma MIN6 cells overexpressing WT, 2S-A and 6S-A Ngn3 were treated with cycloheximide (10μg/ml). Proteins were collected at different time points after treatment and lysed in RIPA-like lysis buffer (50mM Tris-HCl, pH 8; 150mM NaCl; 0.5% NP40 Igepal; 10% Glycerol; protease and phosphatase inhibitor cocktails (Roche; Calbiochem)). Proteins were separated on SDS-PAGE and immmunoblotted with rat anti-HA-Peroxidase (1:1000; Roche) and mouse anti-tubulin (1:1000, Sigma). Protein levels were quantified with ImageJ software (n=3). Half-lives were calculated using first-order kinetics (Prism).
3
C. elegans Developmental Proteome Analysis
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C. elegans were synchronized at 20 °C by bleaching gravid adult animals and maintaining starved L1 larvae for at least 24 h before plating on OP50. For sample collection, animals were harvested after 2 h (L1), 12 h (L2), 32 h (L3), 50 h (L4), and 72 h (gravid adult) on OP50. Number of animals loaded per lane was normalized for actin—~1000 L1s, 800 L2s, 600 L3s, 400 L4s, and 200 gravid adults. Proteins were resolved on 4–12% Bis-Tris polyacrylamide gels (Thermo Fisher), transferred to nitrocellulose membranes (Thermo Fisher), and probed with rat anti-HA-peroxidase 1:1000 (Roche 12013819001), mouse anti-FLAG 1:1000 (Sigma, F1804), mouse anti-actin 1:10,000 (Abcam ab3280), or rabbit anti-CSR-1 1:2000 antibodies8 (link). Secondary HRP antibodies were purchased from Thermo Fisher. Unedited western blots provided in the Source Data File.
4
Analyzing SCA3 Protein Aggregation and Eye Viability
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For examining HA-tagged SCA3polyQ27 or SCA3polyQ78 levels and aggregation and their effect on eye viability (using CD8-GFP), 2-day-old flies were used. At least 30 fly heads per genotype were collected, lysed in Laemli buffer by sonification, and boiled at 95 °C for 5 min. Samples were separated on 12.5% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Fisher Scientific). After blocking in 5% (w/v) non-fat dried milk in PBST for 1 h, the membrane was incubated with primary antibody overnight at 4 °C. The following are antibodies were used: rat anti-HA-Peroxidase (Roche Diagnostics GmbH, Germany), rabbit anti-GFP (A11122; Invitrogen) mouse anti-RFP (6G6 ChromoTek) 6E10 Ab was purchased from Covenance and mouse anti-alpha tubulin from Sigma (T5138). After incubation with secondary antibody, ECL (Amersham) signal was detected in a ChemiDocTM Touch (Bio-Rad). The intensity of the bands was analyzed by using ImageJ (National Institutes of Health, USA). Quantification of western blots was of at least three independent experiments unless indicated otherwise, *p < 0.05; **p < 0.01.
5
Analyzing Ngn3 Isoforms in Pancreatic Organoids
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Pancreatic organoids were infected with GFP-WT-Ngn3-HA or GFP-6S-A-Ngn3-HA and treated with doxycycline (1 μg/μl) for 2 days. Infected GFP+ cells were FACS sorted and lysed in RIPA-like lysis buffer (50 mM Tris-HCl, pH 8; 150 mM NaCl; 0.5% NP40 Igepal; 10% Glycerol, supplemented with protease and phosphatase inhibitor cocktails (Roche; Calbiochem)). Proteins were separated on SDS-PAGE and immmunoblotted for rat anti-HA-Peroxidase (1:1000; Roche) and mouse anti-tubulin (1:1000, Sigma).
6
Temporal Regulation of Ngn3 Phosphorylation
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In vitro translated (IVT) radiolabelled proteins were incubated with interphase (I) or mitotic (M) Xenopus egg extracts. HA-tagged WT and 6S-A Ngn3 proteins were collected 24 hours post transfection and analysed by SDS-PAGE, using rat anti-HA-Peroxidase (1:1000; Roche) and mouse anti-tubulin (1:1000, Sigma). CyclinB Δ90 was produce in E. Coli, purified and incubated with IVT Ngn3 at 21°C for 40 minutes. Endogenous Ngn3 from E14 mouse pancreas was detected with rabbit anti-Ngn3 antibody (1:600; kind gift from Dr Helena Edlund, Figures 1 F and S1 C) or mouse anti-Ngn3 (1:500, Developmental Studies Hybridoma Bank, Figure S2 B). Phosphatase treatment was performed by 30-minute incubation at 30°C with 400 units of Lambda Protein Phosphatase (NEB). Pancreatic ductal mPAC cells expressing HA-tagged WT-Ngn3 were treated with Roscovitine (Selleckchem) and Palbociclib (Selleckchem) at different concentrations for 24 hours.
7
Immunoprecipitation of Flag-tagged Proteins
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Cells expressing indicated constructs were grown to confluency in 10-cm dishes. 48 hr after transfection, cells were lysed in 0.2% Triton X-100, 200 mM NaCl, 50 mM Tris pH 7.4, and 1 mM CaCl2 supplemented with a standard protease inhibitor mixture (Roche Applied Science) and cleared by centrifugation (12,000×g for 10 min). Samples were pre-cleared by incubation with mouse IgG agarose (Sigma) at 4°C for 30 min. Samples were incubated with anti-Flag M2 affinity gel IgG (Sigma) at 4°C for 1 hr, washed with lysis buffer five times, and incubated with SDS sample buffer (Invitrogen) supplemented with dithiothreitol to elute proteins. Western immunoblot analysis was performed using rat anti-HA-peroxidase (Roche) or rabbit anti-Flag (Sigma). Immunoprecipitation signals were quantified by scanning densitometry of films exposed in the linear range. Linearity was verified by generating a standard curve using a dilution series of the indicated sample.
8
Western Blot Analysis of C. elegans Proteins
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Synchronized adult C. elegans were harvested (~72 h at 20 °C after L1 arrest) and 200 adults were loaded per lane. Proteins were resolved on 4–12% Bis-Tris polyacrylamide gels (Thermo Fisher, NW04122BOX), transferred to nitrocellulose membranes (Thermo Fisher, LC2001), and probed with rat anti-HA-peroxidase 1:1000 (Roche 12013819001), mouse anti-FLAG 1:1000 (Sigma, F1804), rabbit Anti-GFP 1: 500 (Thermo Fisher, A-11122), mouse anti-TY-1 1:2000 (Thermo Fisher, MA5-23513), or mouse anti-actin 1:10,000 (Abcam ab3280), and HRP-labeled anti-mouse IgG Secondary 1:10000 (Thermo Fisher A16078) and anti-rabbit Secondary 1:10000 (Thermo Fisher A16110). Unedited western blots provided in the Source Data File. Western Blot quantification was carried out using ImageJ (version 1.53a).
9
C. elegans Developmental Proteome
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C. elegans were synchronized at 20°C by bleaching gravid adult animals and maintaining starved L1 larvae for at least 24 hours before plating on OP50. For sample collection, animals were harvested after 2 hours (L1), 12 hours (L2), 32 hours (L3), 50 hours (L4), and 72 hours (gravid adult) on OP50. Number of animals loaded per lane was normalized for actin -approximately 1000 L1s, 800 L2s, 600 L3s, 400 L4s, and 200 gravid adults. Proteins were resolved on 4-12% Bis-Tris polyacrylamide gels (Thermo Fisher), transferred to nitrocellulose membranes (Thermo Fisher), and probed with rat anti-HA-peroxidase 1:1,000 (Roche 12013819001), mouse anti-FLAG 1:1,000 (Sigma, F1804), mouse anti-actin 1:10,000 (Abcam ab3280), or rabbit anti-CSR-1 1:2,000 antibodies (Claycomb et al., 2009) (link). Secondary HRP antibodies were purchased from Thermo Fisher.
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