Total protein extraction from EDL and pgWAT was performed as previously described (Milkiewicz et al., 2011 (link)). The following polyclonal primary antibodies were used: Ser473-pAkt, Akt, Ser563-pHSL, HSL and α/β-tubulin (Cell Signaling Technology, #4058, #9272, #4139, #4107 and #2148, respectively) and β-actin (sc-47778, Santa Cruz Biotechnology). Secondary antibodies were goat anti-rabbit or anti-mouse IgG-horseradish peroxidase (Jackson ImmunoResearch Laboratories, # 111-035-003, 115-035-003, respectively). Membranes were developed using enhanced chemiluminescence (SuperSignalTM Westpico, #34080, ThermoFisher Scientific) and densitometry analysis was performed with ImageJ Analysis Software (NIH).
Supersignal west pico
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan, United Kingdom, France, Denmark, Canada, China
SuperSignal West Pico is a chemiluminescent substrate used for the detection and quantification of proteins in western blotting applications. It provides a sensitive and reliable method for visualizing protein targets.
Automatically generated - may contain errors
Lab products found in correlation
Femto chemiluminescent substrate, by Thermo Fisher Scientific (68 mentions)
Pvdf membrane, by Merck Group (47 mentions)
Protease inhibitor cocktail, by Roche (24 mentions)
β actin, by Merck Group (23 mentions)
Nitrocellulose membrane, by Bio-Rad (23 mentions)
Immobilon p, by Merck Group (21 mentions)
Supersignal west femto, by Thermo Fisher Scientific (16 mentions)
Whatman, by Cytiva (16 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Bca assay, by Thermo Fisher Scientific (14 mentions)
Hybond, by Cytiva (13 mentions)
Protease inhibitor cocktail, by Merck Group (13 mentions)
Dura chemiluminescent substrate, by Thermo Fisher Scientific (12 mentions)
Las 3000, by Fujifilm (11 mentions)
Protran, by Cytiva (11 mentions)
Pvdf membrane, by Bio-Rad (11 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (11 mentions)
Ripa buffer, by Merck Group (11 mentions)
Trans blot turbo transfer system, by Bio-Rad (11 mentions)
Gapdh, by Cell Signaling Technology (9 mentions)
704 protocols using supersignal west pico
1
Protein Extraction and Western Blot Analysis
2
Quantitative Proteomic Analysis of Extracellular Vesicles
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After isolation, EV samples were resuspended in RIPA lysis buffer. RBC ghosts and human platelet lysates were prepared as in (Prausnitz et al., 1993 (link); Octave et al., 2021 (link)) respectively. Equal protein amounts (except for plasma samples and platelet lysates) were diluted in a buffer containing 10 mM of dithiothreitol (DTT) and then loaded for sodium dodecylsulfate polyacrylamide gel electrophoresis (Mini-Protean TGX Precast Gels 4%–15% (w/v) SDS-PAGE; BioRad or Novex 4–12% Tris-Glycine Gels, Invitrogen). Then, proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked for 2 h. Membranes were incubated overnight with anti-apolipoprotein B100 (Apo B100; BioConnect, SC-13538; 1:500), anti-apolipoprotein A1 (Apo A1; BioConnect, SC-376818; 1:500), anti-CD41 (Abcam, ab134131; 1:2,000), anti-glycophorin A (GPA; Merck, MABF758; 1:1,000), anti-flotillin 1 (BD Biosciences, BD610820; 1:500), anti-ankyrin (Merck, MAB1683; 1:1,000), anti-spectrin (α and β) (Merck, S3396; 1:500), anti-stomatin (Abcam, ab67880; 1:500) or anti-band3 (Invitrogen, MA1-20211; 1:4,000) antibodies. Secondary peroxidase-conjugated goat anti-rabbit or anti-mouse IgGs were then incubated for 1 h and washed. Signal revelation was performed with SuperSignalTM West Pico or Femto (ThermoScientific).
3
Quantitative Proteomic Analysis of Extracellular Vesicles
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After isolation, EV samples were resuspended in RIPA lysis buffer. RBC ghosts and human platelet lysates were prepared as in (Prausnitz et al., 1993 (link); Octave et al., 2021 (link)) respectively. Equal protein amounts (except for plasma samples and platelet lysates) were diluted in a buffer containing 10 mM of dithiothreitol (DTT) and then loaded for sodium dodecylsulfate polyacrylamide gel electrophoresis (Mini-Protean TGX Precast Gels 4%–15% (w/v) SDS-PAGE; BioRad or Novex 4–12% Tris-Glycine Gels, Invitrogen). Then, proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked for 2 h. Membranes were incubated overnight with anti-apolipoprotein B100 (Apo B100; BioConnect, SC-13538; 1:500), anti-apolipoprotein A1 (Apo A1; BioConnect, SC-376818; 1:500), anti-CD41 (Abcam, ab134131; 1:2,000), anti-glycophorin A (GPA; Merck, MABF758; 1:1,000), anti-flotillin 1 (BD Biosciences, BD610820; 1:500), anti-ankyrin (Merck, MAB1683; 1:1,000), anti-spectrin (α and β) (Merck, S3396; 1:500), anti-stomatin (Abcam, ab67880; 1:500) or anti-band3 (Invitrogen, MA1-20211; 1:4,000) antibodies. Secondary peroxidase-conjugated goat anti-rabbit or anti-mouse IgGs were then incubated for 1 h and washed. Signal revelation was performed with SuperSignalTM West Pico or Femto (ThermoScientific).
4
Quantitative CXCL12 Protein Analysis
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Purified CXCL12 and CXCL12 Ch/DS NC were resolved by 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, USA) and electro transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Burlington, EUA). After blocking with 5% bovine serum certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted July 31, 2019. ; https://doi.org/10.1101/616144 doi: bioRxiv preprint albumin (BSA; Sigma), the PVDF membrane was sequentially probed with specific primary antibodies (1:1000, anti-CXCL12, rabbit Ig G, Abcam, Cambridge, UK) and HRP-conjugated secondary antibodies (1:5000, Santa Cruz, Dallas, USA.). The blots were then visualized using the SuperSignal TM West Pico (Thermo Fischer) enhanced chemiluminescence detection system and recorded using a Gel Documentation 2000 system (Bio-Rad).
The copyright holder for this preprint (which was not this version posted July 31, 2019. ; https://doi.org/10.1101/616144 doi: bioRxiv preprint albumin (BSA; Sigma), the PVDF membrane was sequentially probed with specific primary antibodies (1:1000, anti-CXCL12, rabbit Ig G, Abcam, Cambridge, UK) and HRP-conjugated secondary antibodies (1:5000, Santa Cruz, Dallas, USA.). The blots were then visualized using the SuperSignal TM West Pico (Thermo Fischer) enhanced chemiluminescence detection system and recorded using a Gel Documentation 2000 system (Bio-Rad).
5
Quantitative Western Blotting Analysis
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Protein extracts from Drosophila larval brains were obtained by dissecting 10 larval brains in 0.7% NaCl and homogenizing them in 20 µl of 2X Laemmli buffer. Protein samples were loaded into a 4-20% Mini-PROTEAN TGX precast gel to perform electrophoresis (SDS-PAGE) and blotted using the Trans-Blot® Turbo TM Transfer System on a nitrocellulose membrane (Hybond ECL, Amersham). Filters were blocked in 5% non-fat dry milk dissolved in 0.1% Tween-20/PBS for 30 min at RT and, then, incubated with anti-Giotto (1:5000; Rabbit; 59 ) and anti-γH2Av (1:1000; Mouse; Developmental Studies Hybridoma Bank, IA 52242) overnight at 4°C. The membranes were then incubated with HRPconjugated anti-Mouse and anti-Rabbit IgGs secondary antibody (1:5000; Amersham) for 1 hour at RT and then washed again 3 times with 0.1%Tween-20/PBS. The chemiluminescent signal was revealed through either SuperSignal TM West Femto or SuperSignal TM West Pico substrate (Thermo Scientific TM ) using the ChemiDoc scanning system (Bio-Rad). Band intensities were quantified by densitometric analysis using the Image Lab 4.0.1 software (Bio-Rad). WB was repeated independently at least three times.
6
Immunoblotting Assay for Whole Cell Lysates
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Immunoblotting assays for whole cell lysates of cells and tissues were performed as previously described (Tarrago et al., 2018 (link)). Tissues or cells were homogenized and lysed in NETN buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40) supplemented with 50 mM β-glycerophosphate, 5 mM NaF and a protease inhibitor cocktail (Roche). After 30 min of incubation at 4°C, the samples were centrifuged at 12,000 r.p.m. for 10 min at 4°C. Protein concentrations in the supernatants were determined by Bio-Rad protein assay. Lysates were separated by SDS–PAGE, and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). Enhanced chemiluminescence detection was performed using Super-Signal West Pico or Femto Chemiluminescence Substrate (Thermo Scientific). Films were scanned and densitometry was performed using ImageJ. The following antibodies and their dilutions were used for immunoblotting: mouse CD38 (R&D Systems; AF4947, 1:1,000), CD45 (Abcam; AB40763; 1:1000), Phospholamban (PLN) (Invitrogen; MA3-922; 1:1000), CYP3a4 (Proteintech, 18227-1-AP; 1:1000), actin (Cell Signaling Technology; 8457, 1:5,000) and GAPDH (Cell Signaling Technology; 97166, 1:5,000).
7
Quantitative Proteomic Analysis of Alzheimer's Disease
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Postmortem MFG tissues were homogenized in TBS buffer at a ratio of 1:10 (wt/vol) with Pierce Protease and Phosphatase Inhibitor Cocktail (A32965, ThermoScientific) on ice. Tissue lysate was sonicated and then centrifuged at maximum speed for 15 min at 4 °C. Protein concentrations were measured using the BCA protein assay kit (Bio-Rad Laboratories, Inc.). Electrophoresis was performed using 30 μg of protein lysates, resolved in a 4–12% SDS-PAGE gel (CriterionTM TGXTM, Bio-Rad Laboratories, Inc.) and transferred to a nitrocellulose membrane (Immobilon®-P, Millipore) that was blocked with 5% BSA in TBS with 0.01% tween, followed by overnight incubation of primary antibodies; HT7 (1:300, MN1000, Thermo Fisher), AT8 (1:1000, MN1020, Invitrogen) and PHF1 (1:1000, Peter Davies antibodies) diluted in the blocking solution. Horseradish peroxidase (HRP) secondary antibodies (goat anti-mouse HRP conjugated (1:10,000, 626820, Invitrogen) were incubated for 2 h at RT and the proteins were detected with Supersignal West Pico (34580, Thermo Scientific) and imaged by using iBright 1500 (Invitrogen). Western blots were analyzed using ImageJ Fiji.
8
Protein Analysis via Immunoblotting
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For protein analysis, samples were analyzed by SDS-PAGE, transferred to nitrocellulose membranes, and blocked with 3% bovine serum albumin (BSA) for immunoblot analysis. Detection with horseradish peroxidase was performed using SuperSignal West Pico (Thermo Fisher Scientific). Polyclonal and monoclonal antibodies used in this study are described in detail elsewhere (FabD and Ino1 are described in reference 11 (link); Log in reference 15 (link); Pup in reference 6 (link)).
9
Western Blot Analysis of Intestinal Proteins
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Tissue samples were stored at ‒80 °C and homogenized in NETT buffer (150 mM NaCl + 5 mM EDTA + 10 mM Tris + 1% TritonX-100 in deionized H2O) with protease inhibitor. Small intestine samples were divided into six equal sections, and sections corresponding to the jejunum were used for lysis. Unfortunately, some small intestine samples degraded during lysis, so only undegraded samples were used for western blot analysis. Protein concentrations of the collected lysates were analyzed with the RC DCTM Protein Assay (Bio-Rad Life Science, Hercules, CA, USA). 100–120 μg of protein from tissue lysates were separated by gel electrophoresis in a 10% SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes (GVS, Sanford, ME, USA). Proteins were probed using our custom rabbit anti-mouse primary antibodies, a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (NA9340; GE Healthcare, Chicago, IL, USA), and an HRP-conjugated GAPDH antibody for loading control (HRP-60004; Proteintech, Rosemont, IL, USA). Antibodies were diluted (ZIP14 1:2000, ZnT10 1:1000) in blocking buffer (5% Non-fat dried milk in TBST (Tris-buffered saline + Tween 20)). An enhanced chemiluminescent substrate (SuperSignal West Pico; Thermo Fisher Scientific, Waltham, MA, USA) was used for signal detection with the ChemiDoc™ MP Imaging System (Bio-Rad Life Science).
10
Western Blot Analysis of CMV Proteins
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Cells were lysed with RIPA buffer (0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA, 150 mM NaCl, and 10 mM Tris at pH 7.2) containing protease inhibitor cocktail (Roche). Ten micrograms of the total lysate was separated in 10% SDS-polyacrylamide gels and transferred to PVDF membranes (Millipore). Primary antibodies used in this paper are mouse anti-CMV IE1/2 monoclonal antibody (MAB8131, Millipore), mouse anti-CMV pp52 monoclonal antibody (CH16, Santa Cruz Biotechnology), mouse anti-CMV pp28 monoclonal antibody (CH19, Santa Cruz Biotechnology), rabbit anti-ZAP polyclonal antibody (PA5-31650, Invitrogen), mouse anti-TRIM25 monoclonal antibody (BD Biosciences), rabbit anti-T7 tag monoclonal antibody (D9E1X, Cell Signaling Technology), mouse anti-alpha tubulin monoclonal antibody (DM1A, Abcam) and mouse anti-β-Actin monoclonal antibody (Abcam). Blots were probed with primary antibody (1:500–1:5000) diluted in 5% dehydrated milk in Tris Buffered Saline (TBS) and subsequently the HRP-conjugated secondary antibodies (Pierce) at 1:5000. Blots were washed in TBS three times, incubated with chemiluminescent substrate (SuperSignal West Pico; Thermo Scientific) according to the manufacturer’s protocol, and exposed in G:Box (Syngene) for visualization of bands.
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