Brain tissue was dissected from E10, E12, P0, and P42 mice. Whole brains were used for the analysis of embryonic tissues and half brains cut mid-sagittally were used for postnatal tissue analysis. Protein quantification was performed using Bio-Rad DC protein assay kit and Perkin Elmer Wallac 1420 multilabel counter. Primary antibody for Ferritin was anti-Ferritin Heavy Chain (FTH1) polyclonal rabbit antibody #3998 from Cell Signaling and was used at 1:1000 for 4 days in a cold room. Secondary antibody was goat anti-rabbit IgG conjugated to HRP (Invitrogen G21234). Ferritin protein has a molecular weight of 21 kDa and beta-actin is 42 kDa. Blots were cut at 35 kDa. Beta-actin antibody was mouse monoclonal IgG1 conjugated to HRP (Santa Cruz Biotechnology sc-47778 HRP).
Sc 47778 hrp
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-47778 HRP is a horseradish peroxidase (HRP) conjugate product from Santa Cruz Biotechnology. HRP is an enzyme used as a label or detection reagent in various immunoassay and detection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Sds page gel, by Thermo Fisher Scientific (4 mentions)
Ab32072, by Abcam (4 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (3 mentions)
Complete mini, by Roche (3 mentions)
Hrp conjugated β actin antibody, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Ab71435, by Abcam (2 mentions)
Ab54481, by Abcam (2 mentions)
Wh0008424m1, by Merck Group (2 mentions)
Sab1403807, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
A1978, by Merck Group (2 mentions)
Ab179466, by Abcam (2 mentions)
Ab62352, by Abcam (2 mentions)
Mabc1640, by Merck Group (2 mentions)
Ab187091, by Abcam (2 mentions)
Mabc604, by Merck Group (2 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab109497, by Abcam (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
31 protocols using sc 47778 hrp
1
Ferritin Quantification in Developing Mouse Brain
2
Immunoblotting Analysis of TSPO
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After stimulation of TLR ligands, cells were lysed in 1% Triton X-100 lysis buffer (1% Triton X-100, 250 mM sucrose, 20 mM Tris-HCl (pH 7.2), 1 mM EDTA (pH 8.0), 1 mM phenylmethylsulfonyl fluoride, 50 mM NaCl) with 1× protease and phosphatase inhibitors and then cell lysate were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. Membranes were incubated with antibodies specific for TSPO (ab109497, Abcam, Cambridge, UK) or β-actin HRP (sc-47778 HRP, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bound TSPO antibody was detected by species-specific, horseradish peroxidase-conjugated secondary antibody. Chemiluminescence detection was performed to analyze the protein bands of interest.
3
VASP Protein Expression Analysis in Bladder Cancer
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BCa tissue specimens were collected in Germany (Department of Urology and Urological Oncology, Hannover Medicine School) from patients undergoing resection of the bladder. All individuals gave written informed consent. All experimental protocols for tissue sample collection were approved by the Hannover Medical School Ethics committee (case number: 614–2009) and experiments were performed according to relevant guidelines. Specimens from non-muscle invasive (n = 3), muscle invasive (n = 3) BCa and negative biopsies (n = 3) were analyzed. Tissue lysis was performed as described earlier49 (link). 20 μg of total protein per extract were separated by NuPAGE® Gradient Gel 4–12% under reducing conditions and electroblotted to nitrocellulose membrane (LG), as presented elsewhere60 (link). Membranes were incubated overnight at 4 °C with the primary mouse anti-VASP antibody (Enzo LifeScience, ALX-804-177-C050, dilution 1:500) or anti- β-actin antibody conjugated to HRP (Santa Cruz, sc-47778 HRP, 1:4,000), in the first case followed by incubation with anti-mouse HRP-conjugated secondary antibody (Santa Cruz; dilution 1:2,000) for 2h at room temperature. Target protein was detected by Enhanced Chemiluminescence (Perkin-Elmer LAS, Inc.).
4
Western Blot Antibody Reagents
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Rabbit anti-cytosolic PEPCK was prepared in our laboratory [18 (link)]. Goat anti-muscle pyruvate kinase (PK-M) was from Rockland Immunochemicals Inc. (Gilbertsville, PA). An HRP-conjugated β-actin antibody was from Santa Cruz Biotechnology (sc-47778-HRP; Santa Cruz, CA). Rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) was from Cell Signaling Technology Inc. (#4370, Beverly, MA). Secondary antibodies were goat anti-rabbit IgG-HRP, rabbit anti-goat IgG-HRP (Jackson ImmunoResearch, West Grove, PA), and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, OR).
5
Western Blotting Protocol for Protein Detection
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Western transfer was performed in Tris–Glycine transfer buffer to a nitrocellulose membrane (0.45 µm pore, Pall Scientific, Port Washington, New York)83 (link). Membranes were blocked in 1% milk (α-tubulin) or 5% BSA (β-Actin) for 1 h. Primary antibodies (mouse anti-α tubulin, Sigma T6074 1:5000; mouse anti-β actin HRP, Santa Cruz sc-47778 HRP, 1:5000, Dallas, Texas) were incubated at 4 °C overnight with gentle shaking. Membranes were washed with TBS 0.1% Tween-20 (TBST) and tubulin blots were probed with anti-mouse secondary (Pierce 31,430, Rockford, Illinois) for 1 h. Actin blots did not require a secondary antibody, as mouse anti-β actin is already HRP conjugated. Membranes were developed using Premium Western Blotting Reagent (LI-COR, Lincoln, Nebraska) and scanned for chemiluminescence on a C-Digit Scanner (LI-COR).
6
Immunoblotting for Protein Expression
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Immunoblot analysis was performed as previously described (11 (link)). Proteins of interest were detected using antibodies specific for rabbit TRPC6 (1:1000, Cell Signaling #16716S), rabbit cPLA2 (1:1000, Cell Signaling #2832), mouse iPLA2 (1:1000, Santa Cruz Biotechnology #sc-376563), and β-actin (1:2000, Santa Cruz Biotechnology #sc47778 HRP). Anti-rabbit (1:1000, antibodies-online #ABIN102010) or anti-mouse (1:1000, Santa Cruz Biotechnology #SC516102) antibodies were used for secondary antibodies.
7
Western Blot Analysis of Cell Signaling
8
Protein Extraction and Western Blot Analysis
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Proteins were extracted using RIPA buffer: 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5% Na-deoxycholate, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, and proteinase (Roche) plus phosphatase (Roche) inhibitor cocktails. Protein extracts were resolved using 4–12% SDS-PAGE gels (Life Technologies) and transferred to nitrocellulose membranes (Life Technologies). Membranes were probed with primary antibodies overnight on 4 °C shaker, then incubated with horseradish-peroxidase conjugated secondary antibodies and signals were visualized with ECL (Bio Rad). The primary antibodies used are as follows: β-Actin (Actin) (sc-47778 HRP, Santa Cruz, 1:10,000), PGC1α (ab54481, Abcam, 1:500), BBOX1 (WH0008424M1, Sigma Aldrich, 1:500), CPT2 (ab71435, Abcam, 1:500), FASN (SAB1403807, Sigma Aldrich, 1:1000), pACC1/2 (11818, Cell Signaling, 1:1000), ACC1 (4190, Cell Signaling, 1:1000), ACC2 (8578, Cell Signaling, 1:1000), AMPK (2532, Cell Signaling, 1:1000), pAMPK (2535, Cell Signaling, 1:1000) and c-MYC (MYC) (ab32072, Abcam, 1:1000).
9
Western Blot Analysis of ATP7B Protein
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Cells were homogenized in RIPA lysis buffer (60 mM tris-HCl, 150 mM NaCl, 2% Na-deoxycholate, 2% Triton X-10, 0.2% SDS, and 15 mM EDTA) and protease inhibitors (Roche, Basel, Switzerland; Complete Mini, EDTA-free). 10 μg protein lysate of whole protein extract was separated on a 10% SDS gel. Samples were blotted onto PVDF membranes and blocked with 5% semi-skimmed milk powder (Roth, Karlsruhe, Germany) in Tris-buffered saline supplemented with 0.1% Tween 20 (Merck, Darmstadt, Germany). For detection of ATP7B protein (ATPase7), a monoclonal rabbit anti-human ATP7B antibody (1:1,000, ab124973, Abcam, Cambridge, UK) was added overnight. β-Actin was assessed for protein loading control (1:1,000, sc-47778 HRP, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibody was horseradish peroxidase-conjugated anti-rabbit (1:10,000; GE Healthcare life science, München, Germany) and incubated 2 hours at room temperature. For detection, ECL Western Blotting Detection Reagent (GE Healthcare) was added and the membrane was exposed to film (Hyperfilm, GE Healthcare).
10
Western Blot Analysis of Synovial Proteins
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Synovium tissue or synovial fibroblasts were homogenized with radioimmunoprecipitation assay buffer containing protease inhibitor cocktails (PICs) and phenylmethanesulfonylfluoride (phenylmethylsulfonyl fluoride [PMSF]) (Sigma-Aldrich) on ice. Equal amounts of protein (30 μg) from the different groups were denatured in SDS loading buffer and separated on 10% SDS-PAGE gels. Proteins were transferred to NC membranes in the transfer buffer containing 20% methanol. Membranes were sequentially blocked with 5% skim milk, incubated with the primary antibodies described below overnight (4°C), and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000, Jackson ImmunoResearch) at room temperature for 1 h. Signals were analyzed using an enhanced chemiluminescence western blot system (Bio-Rad Laboratories). β-Actin (1:3000, sc-47778 HRP; Santa Cruz) was used as the internal control. The following antibodies were used: anti-RGS12 (1:1,000; GW21317; Sigma-Aldrich), anti-KIF2A (1:1,000; 13105-1-AP; Proteintech), and anti-MYCBP2 (1:1,000; MABN2397; Sigma-Aldrich).
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