To demonstrate expression of αIFNα-ib in D1-αIFNα-ib-eGFP cells, 106 cells were incubated in 100 μl Cytobuster protein extraction reagent (Merck) for 5 minutes, centrifuged and heat denatured. 20 μl were analysed by 12% SDS-PAGE and Western blotting. Protein blotting was performed via semi-dry blot onto a PVDF membrane using 48 mM Tris, 39 mM glycine for 30 min at 15 V. After protein transfer, the membrane was washed for 10 min in TBST and blocked for 1 h at room temperature with 3% skimmed milk. Blots were treated for 1 h with mouse anti-myc antibody (9E10, Santa Cruz, 1:2500), washed 3x with TBST, followed by 1 h with goat anti-mouse IgG-Fc alkaline phosphatase labelled antibody (Promega, 1:7500). After washing 3x with TBST and once with AP buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2), blots were developed with BCIP/NPT-substrate (66 μl NBT and 33 μl BCIP in 10 ml AP-buffer, Promega).
Cytobuster protein extraction reagent
Manufactured by Merck Group
Sourced in Germany, United States, United Kingdom, Switzerland
CytoBuster Protein Extraction Reagent is a cell lysis buffer designed for the efficient extraction and solubilization of proteins from various cell types. It is a non-ionic detergent-based solution that effectively disrupts cell membranes and releases intact proteins for further analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (8 mentions)
Clarity western ecl substrate, by Bio-Rad (8 mentions)
Protease and phosphatase inhibitor cocktail tablet, by Roche (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Amersham imager 600, by GE Healthcare (5 mentions)
Amersham imager, by Cytiva (5 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Kaleidoscope protein ladder, by Bio-Rad (3 mentions)
Ecl plus western blotting detection reagent, by GE Healthcare (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Chemi lumi one super, by Nacalai Tesque (3 mentions)
Protease inhibitor cocktail tablet, by Roche (2 mentions)
Ab9485, by Abcam (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Avantor (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
61 protocols using cytobuster protein extraction reagent
1
Detecting αIFNα-ib Expression in D1-αIFNα-ib-eGFP Cells
2
Protein Extraction and Western Blotting for PLD2 Analysis
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Epithelial cell lysates were prepared using Cytobuster Protein Extraction Reagent (71009, EMD Millipore) with protease inhibitors (cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets, Roche) and PhosSTOP (Roche 04906837001). Cytoplasmic and nuclear extracts were prepared using Nucbuster (EMD Millipore 71183) following the manufacturer’s protocol. SDS-PAGE and western blotting were as described elsewhere.48 (link) Primary antibodies were a rabbit anti-PLD2 antibody (PLD2-26, Denmat-Ouisse et al., 2001) used at 1:1000 and a rabbit anti-GAPDH polyclonal (Abcam ab9485) used at 1:10,000. The secondary antibody, a horse radish peroxidase (HRP)-linked anti-rabbit IgG (Cell Signalling, 7074S), was used at 1:10,000. A kaleidoscope protein ladder (Bio-Rad, 1610375) was used throughout.
3
Western Blotting of Transfected Cell Lysates
4
TACE Activity Assay Protocol
5
Western Blot Analysis of PLD1 in Epithelial and Stromal Cells
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Epithelial cell lysates were prepared using Cytobuster Protein Extraction Reagent (71009, EMD Millipore, Watford, UK) with protease inhibitors (cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets, Roche, Burgess Hill, UK). Stromal cell lysates were prepared in a 1% TX-100 lysis buffer containing protease inhibitors. SDS-PAGE and western blotting were as described previously (Rumsby et al, 2011 (link)). Primary antibodies were an anti-PLD1 rabbit polyclonal (sc25512, Santa Cruz, Insight Biotechnology, Wembley, UK, 1 : 750), and a rabbit anti-GAPDH polyclonal (Abcam, Cambridge, UK, ab9485, 1 : 20 000). The secondary antibody was HRP-linked anti-rabbit IgG (7074S, Cell Signalling, New England Biolabs Ltd, UK, 1 : 6000). A kaleidoscope protein ladder (Bio-Rad, Watford, UK, Cat No 1610375) was used throughout.
6
Famitinib Modulates Protein Expression
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Total proteins prior and subsequent to famitinib treatment were extracted from BGC-823 and MGC-803 cell pellets using CytoBuster Protein Extraction Reagent (Merck Millipore, Darmstadt, Germany). Proteins were quantified with a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.), and ~20 µg of protein was separated on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were then transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Chalfont, UK), which was subsequently incubated with anti-cyclin B1 (dilution, 1:1,000; catalog no., AJ1208a; Abgent Inc., San Diego, CA, USA), rabbit polyclonal anti-B-cell lymphoma 2 (BCL2; dilution, 1:1,000; catalog no., 2872; Cell Signaling Technology, Inc., Danvers, MA, USA) and mouse monoclonal anti-β-actin (dilution, 1:3,000; catalog no., A5441; Sigma-Aldrich, St. Louis, MO, USA) antibodies at 4°C overnight. Secondary anti-rabbit and anti-mouse horseradish peroxidase-conjugated IgG antibodies (dilution, 1:3,000; catalog nos., 7074 and 7076, respectively; Cell Signaling Technology, Inc.) were applied and allowed to incubate at room temperature for 1 h. Proteins were visualized with ECL Plus Western Blotting Detection Reagent (GE Healthcare Life Sciences).
7
Insulin-degrading enzyme activity assay
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Liver tissue extracts were prepared by homogenizing tissue in Cytobuster Protein Extraction Reagent (Cat. 71009-3, EMD Millipore, Billerica, MA 01821) according to the manufacturer’s recommended protocol. IDE activity was assessed with the InnoZyme Insulysin/IDE Immunocapture Activity Assay Kit (Cat. CBA079, Calbiochem/Millipore) and was normalized to the value of control group. Relative IDE activity was expressed in this study.
8
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cell pellets using CytoBuster Protein Extraction Reagent (Merck Millipore, Darmstadt, Germany). Protein concentration was measured by using a BCA Protein Assay Kit (Beyotime Biotechnology, Jiangsu, China), and 30 μg of protein from each sample was separated by 12 % SDS-PAGE. After transfer, the nitrocellulose membrane (GE Healthcare, Piscataway, NJ) was incubated with the corresponding primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h (the antibody list is shown in Additional file 1 : Table S1). Proteins were visualized using ECL plus Western Blotting Detection Reagents (GE Healthcare).
9
Western Blot Analysis of BRF1, BRF2, and Actin
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Total cellular protein was extracted using Cytobuster Protein Extraction Reagent (Merck Millipore; 71009). Proteins were fractionated by SDS-PAGE and transferred to nitrocellulose membranes, which were incubated overnight with antibodies against BRF1 (Bethyl Laboratories; A301-228A), BRF2 and actin (Santa Cruz Biotechnology; sc-390312 and sc-1615). Membranes were then incubated with HRP-conjugated anti-goat (Dako; P0449) anti-rabbit and anti-mouse (Cell Signaling; 7074 and 7076) IgG for 1 h. Bands were visualized using the enhanced chemiluminescence method.
10
Western Blot Protein Expression Analysis
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Cells were lysed using a CytoBuster protein extraction reagent (Merck Millipore, Darmstadt, Germany) in the presence of protease and phosphatase inhibitor cocktail tablets (Roche, Basel, Switzerland). Protein concentration was measured by a BCA Protein Assay Kit (Beyotime, Jiangsu, China). Soluble lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Merck Millipore). After blocking with 5% bull serum albumin (Amresco, Solon, OH) or fat-free milk, membranes were probed with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. Signals were visualized using Amersham Imager 600 (GE Healthcare, Chicago, IL) after incubation with Clarity Western ECL substrate (Bio-Rad, Hercules, CA). Protein expressions were quantified using ImageJ Version 1.48 software and normalized to β-actin level followed by calculations of relative ratios to controls.
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