Total protein lysates were prepared with RIPA buffer containing protease and phosphatase inhibitors. Protein extracts were clarified, denatured, and subjected to SDS-PAGE and Western blotting. The primary antibodies employed were: rabbit polyclonal anti-CIP2A (Sigma); rabbit monoclonal anti-AKT, rabbit monoclonal anti-pAKT (Cell Signaling Technology); and mouse monoclonal anti-β-actin (Sigma). All antibodies were used at 1:1000 except β-actin, which was used at 1:5000. Proteins were detected with the appropriate secondary antibodies conjugated to peroxidase (HRP, GE Healthcare) by chemiluminescence using Immobilon Crescendo Western HRP substrate (Merck Millipore, Burlington, MA, USA).
Immobilon crescendo western hrp substrate
Manufactured by Merck Group
Sourced in United States, Germany
Immobilon Crescendo Western HRP substrate is a chemiluminescent substrate used for the detection of horseradish peroxidase (HRP) in Western blot applications. It provides a bright, stable signal for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Amersham imager 600, by GE Healthcare (10 mentions)
Anti rabbit igg hrp, by Merck Group (9 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Anti gapdh, by Santa Cruz Biotechnology (7 mentions)
Protease inhibitor, by Merck Group (7 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Anti s2, by Sino Biological (4 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
Anti mouse igg peroxidase, by Merck Group (3 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
β actin, by Merck Group (2 mentions)
C digit blot scanner, by LI COR (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Anti β actin, by Merck Group (2 mentions)
61 protocols using immobilon crescendo western hrp substrate
1
Protein Lysate Preparation and Analysis
2
Quantitative Analysis of Tight Junction Protein ZO-1
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Western blot analysis was done according to Schwarzer et al., (2016 (link)). Frozen colon samples were homogenized in T-PER (tissue protein extraction, Thermo Scientific, IL, United States) supplemented with protease inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail, Thermo Scientific, IL, United States). After centrifugation of the lysates, protein concentration was estimated in the supernatant using a PierceTM BCA Protein Assay Kit (Thermo Scientific, IL, United States). Total proteins (4 µg/µl) were separated on 4%–20% precast gels SDS–polyacrylamide gel (Bio-Rad Laboratories, United States) and then transferred to Immobilon-P membranes (PVDF membrane, Merck Millipore, MA, United States). Membranes were blocked with 4% BSA in TBS-Tween for 1 hour at RT before being probed overnight at 4°C with specific primary antibodies against ZO-1 (Invitrogen, CA, United States). After washing, Ab binding was revealed by incubation with anti-rabbit IgG HRP-conjugated antibody (Abcam, Cambridge, United Kingdom) for 1 hour at room temperature, and proteins were visualized by chemiluminiscent (Immobilon Crescendo Western HRP Substrate, Merck Millipore, Germany) substrate according to the manufacturer’s instructions. Signals were measured by C-Digit Blot Scanner (LI-COR, Lincoln, United States) and quantified using the Image Studio Digits, version 3.1 and program ImageJ.
3
Protein Quantification by Western Blot
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Proteins were resolved by SDS-PAGE and transferred to PVDF (Millipore). Membranes were blocked with 10% milk powder followed by incubation with primary antibodies against β-globin (Santa Cruz), Band 3 (IBGRL), MTAP, NF-E2 (both from Abcam) or β-actin (Sigma). Secondary antibodies were goat α-rabbit IgG-HRP and rat α-mouse IgG1-HRP (Abcam). Membranes were incubated with Immobilon Crescendo western HRP substrate (Merck), and bands were visualised by ImageQuant LAS 4000 (GE Life Sciences). For protein quantitation, the intensities of visualised bands were obtained from ImageJ 1.52.
4
Western Blot Protein Analysis Protocol
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Protein samples were separated by SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immobilon-P, EMD Millipore). After blocking with 2% (w/v) bovine serum albumin in TBS-T [50 mM tris-HCl, 150 mM NaCl, and 0.5% (v/v) Tween 20 (pH 7.4)], the membrane was incubated with the primary antibodies diluted in tris-buffered saline [TBS; 50 mM tris-HCl and 150 mM NaCl (pH 7.4)], followed by incubation with horseradish peroxidase (HRP)–conjugated secondary antibodies. The bound primary antibodies were detected using an Immobilon Crescendo Western HRP substrate (EMD Millipore) according to the manufacturer’s instructions. The total protein contents were detected using SYPRO Ruby protein gel stain (Thermo Fisher Scientific) according to the manufacturer’s instructions.
5
Protein Extraction and Western Blot
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Cells were lysed in NP40 lysis buffer (Invitrogen™) containing 1% Halt Protease Inhibitor Cocktail (Thermo Scientific) and 10% PhosSTOP (Sigma-Aldrich). Lysates were cleared and stored at − 80 °C. Protein was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Scientific™). Samples were prepared using Laemmlli buffer (SDS—Sample Buffer, Reducing, 4x; Boston Bioproducts). At least 15 μg of protein per sample was resolved on NuPAGE™ Bis-Tris 4–12% gels (Invitrogen) in NuPage™ MOPS SDS Running Buffer (Invitrogen™). Protein was transferred to the PVDF membrane and blocked in either 3–5% bovine serum albumin (BSA), protease-free (Roche), or non-fat dry milk (NFDM; Cell Signaling). Blots were developed using Pierce ECL Western Blotting Substrate (Thermo Scientific™), Immobilon Crescendo Western HRP substrate (EMD Millipore), or Immobilon Forte Western HRP substrate (EMD Millipore).
6
Western Blot Analysis of Bacterial LTB Protein
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Samples (15 µl) of concentrated
bacterial growth media mixed with 5 µl sample buffer (Bio-Rad) were
separated on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel,
proteins were transferred to a 0.45 µm PVDF membrane (Merck) by
blotting for 7 min using eBlotTM protein transfer system
(Genscript). Membranes were then blocked with 2.5% skim milk in
phosphate buffer saline (PBS; Bio-Prep), for 1 h at room
temperature, on a rotary shaker (50 rpm). Subsequently, the
membrane was washed in PBS supplemented with 0.05% Tween-20 (PBST)
for four 5-min increments with gentle agitation (50 rpm). The
membrane was incubated overnight at room temperature with anti-LTB
antibody (GeneTex) diluted 1:5000, then washed with PBST as
described above, incubated with goat anti-mouse HRP (abcam) diluted
1:10000 in 2.5% skim milk- PBST, and washed again with PBST. The
membrane was then stained for 1 min with 2 ml Immobilon Crescendo
western HRP substrate (Merck Millipore) and visualized using
ImageQuant LAS 4000 multipurpose CCD camera system (GE
Healthcare).
bacterial growth media mixed with 5 µl sample buffer (Bio-Rad) were
separated on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel,
proteins were transferred to a 0.45 µm PVDF membrane (Merck) by
blotting for 7 min using eBlotTM protein transfer system
(Genscript). Membranes were then blocked with 2.5% skim milk in
phosphate buffer saline (PBS; Bio-Prep), for 1 h at room
temperature, on a rotary shaker (50 rpm). Subsequently, the
membrane was washed in PBS supplemented with 0.05% Tween-20 (PBST)
for four 5-min increments with gentle agitation (50 rpm). The
membrane was incubated overnight at room temperature with anti-LTB
antibody (GeneTex) diluted 1:5000, then washed with PBST as
described above, incubated with goat anti-mouse HRP (abcam) diluted
1:10000 in 2.5% skim milk- PBST, and washed again with PBST. The
membrane was then stained for 1 min with 2 ml Immobilon Crescendo
western HRP substrate (Merck Millipore) and visualized using
ImageQuant LAS 4000 multipurpose CCD camera system (GE
Healthcare).
7
Western Blot Analysis of Recombinant Proteins
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For WB analysis, 7 µg of each recombinant protein was separated by 12% SDS-PAGE and then transferred onto nitrocellulose membrane. The membrane was blocked with 5% non-fat skim milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h at room temperature with constant shaking. The membrane was then washed three times with TBS-T and incubated with human serum samples diluted at 1:200 for 1 h. The membrane was then washed again and treated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-human IgG antibodies (Jackson ImmunoResearch, Ely, UK), diluted at 1:100,000. After further washes, the reaction was developed by the addition of chemiluminescence peroxidase substrate (Immobilon Crescendo Western HRP substrate, Merck, Darmstadt, Germany), and results were visualized using Image Lab software (Bio-Rad, Hercules, CA, USA).
8
Western Blot Analysis of SARS-CoV-2 Spike Protein
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Cells in 12-well plates were lysed by 125 μl of radioimmunoprecipitation assay buffer (89901, Thermo Fisher Scientific) with protease inhibitor (4693159001, Roche, Basel, Switzerland) at 24 hours after transfection. Proteins were separated by SDS–polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride or nitrocellulose membrane. Specific primary antibodies were incubated with the blocked membranes at 4°C overnight, followed by horseradish peroxidase (HRP)–conjugated secondary antibodies (Thermo Fisher Scientific) incubation for 2 hours at room temperature. The signal was developed by the Immobilon Crescendo Western HRP Substrate (WBLUR0500, Merck Millipore, MA, USA) and detected using the Alliance Imager apparatus (Uvitec, Cambridge, UK). Mouse anti-flag antibody (F1804, Sigma-Aldrich, St. Louis, Missouri, USA) was used as the primary antibody to detect the overexpression of the flag-tagged proteases. Human ACE2 was detected with a rabbit anti-V5 antibody (CM3005, ImmunoWay, Plano, TX, USA). β-Actin was used as the loading control, which was detected by a mouse anti-human β-actin antibody (MAB8929, R&D Systems, Minneapolis, Minnesota, USA). The spike protein was detected with a rabbit anti–SARS-CoV-2 spike S2 antibody (40590-T62, Sino Biological, Beijing, China).
9
Western Blot Analysis of Cytoskeletal Proteins
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Proteins were extracted in 1× RIPA Buffer (Sigma‐Aldrich) with 1× Protease Inhibitor Cocktail (PIC) and 1× phenylmethylsulfonyl fluoride (PMSF) and thereafter quantified with Bradford protein reagent (Bio‐Rad Laboratories GmbH, Munich, Germany) and resolved (10 µg) in Acrylamide/Bis‐acrylamide gels followed by transference to polyvinylidene difluoride (PVDF) Amersham HybondTM‐P membranes (GE Healthcare) plus 2 h blocking in 1× PBS/5% non‐fat‐dried milk/0.2% Tween‐20 at room temperature. Overnight incubation with antibodies (Cell Signaling Technology) against β‐actin (Cat# 4970, RRID:AB_2223172; 1:1000), CDC42 (Cat# 2466, RRID:AB_2078082; 1:500) and RHOA (Cat# 2117, RRID:AB_10693922; 1:500) in 1× PBS/0.5% non‐fat‐dried milk/Tween‐20 0.2% was performed at 4°C and further incubation with the secondary antibody‐HRP against rabbit (Millipore Cat# AP156P, RRID:AB_11213985; 1:1000) was done for 2 h at room temperature. Immobilon® Crescendo Western HRP Substrate (Merck Millipore) was used, revealed in an Amersham Imager 600 (GE Healthcare). Data were curated using Quantity One 1‐D software (RRID:SCR_014280; Bio‐Rad).
10
Western Blot Analysis of Cellular Proteins
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Cells were lysed with 9 M urea lysis buffer (20 mM HEPES, pH 8.0, 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, and 1 mM β-glycerophosphate) and protein amount were quantitated using Pierce™ 660nm Protein Assay Kit. Samples were then run on 4 - 12% Bis-Tris SDS-PAGE gel (Invitrogen) and transferred to a 0.2 µM PVDF membrane (Bio-Rad Laboratories). The membrane was blocked with 5% non-fat dried milk in TBST (20mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h before incubating with the respective primary antibody in TBST overnight at 4 °C on a roller. Subsequently, the membrane was subjected to washing with TBST for 5 min thrice; incubation with the respective secondary antibody in 5% non-fat dried milk in TBST for 1 h; washing with TBST for 5 min thrice; 1 min incubation with the ECL substrate (Immobilon Crescendo Western HRP substrate, MerckMillipore) and exposed to X-ray film for signal detection. AXL antibody was purchased from R&D systems (AF154) and GAPDH antibody from Santa Cruz Biotechnology (sc-32233). For vitreous analysis, soluble sAPPα fragment detecting antibody was purchased from IBL (11088).
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