Total proteins were extracted using Complete Lysis buffer containing proteases inhibitors (04719956001 Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitors (04906845001 Roche). Protein lysates were separated by NuPage 4–12% Bis-Tris Gel (NP0335BOX Invitrogen) under reducing conditions. Western blotting (WB) was carried out according to standard techniques, with rabbit anti-pATM (phospho S1981) ab81292, anti-ATM ab32420, and anti-b-tubulin (HRP) ab21058 (Abcam, Cambridge, MA, USA). Immunoreactive proteins were detected by ECL Prime (GE Healthcare, RPN2232) and a chemiluminescence gel documentation and analysis system (MINI HD, UVITEC, Cambridge, UK).
Mini hd
Manufactured by Uvitec
Sourced in United Kingdom
The MINI HD is a compact and versatile laboratory equipment designed for various applications. It features a high-definition imaging system capable of capturing detailed images and videos. The MINI HD is suitable for use in a wide range of laboratory settings, providing a reliable solution for visual analysis and documentation tasks.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime, by GE Healthcare (4 mentions)
Phosphatase inhibitor, by Roche (2 mentions)
Complete mini 04693124001, by Roche (2 mentions)
Ab81292, by Abcam (1 mentions)
Ab32420, by Abcam (1 mentions)
Ab21058, by Abcam (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Murine anti phospho stat3 py705 and anti stat3 mabs, by BD (1 mentions)
Rabbit anti socs3, by Cell Signaling Technology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo mini pvdf membrane, by Bio-Rad (1 mentions)
Specific hrp conjugated secondary antibodies, by Merck Group (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Rabbit anti phospho stat1 py701 and anti stat1 anti sera, by Cell Signaling Technology (1 mentions)
α tubulin mab, by Merck Group (1 mentions)
Cleaved caspase 3 rabbit mab, by Cell Signaling Technology (1 mentions)
5 protocols using mini hd
1
Protein Extraction and Western Blotting
2
Western Blot Analysis of JAK-STAT Signaling
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Cells were lysed in lysis buffer (20 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Brij97) containing 2 mM Na Orthovanadate and protease inhibitors (Roche Diagnostics, Complete Mini 04693124001). Lysates were resolved under reducing conditions by SDS-PAGE (10% or 13% acrylamide) and analyzed by Western blotting using the following antibodies: rabbit anti-phospho-STAT1 (pY701) and anti-STAT1 anti-sera (Cell Signaling Technology, 9167 and 9172, respectively), murine anti-phospho-STAT3 (pY705) and anti-STAT3 mAbs (BD Transduction Laboratories, 612,356 and 610,190, respectively), rabbit anti-SOCS3 (Cell Signaling Technology 2932) and murine α-tubulin or β-actin mAbs (Sigma-Aldrich T6074 and A2228, respectively). Proteins were detected by ECL Prime (GE Healthcare, RPN2232) and visualized by a chemiluminescence gel documentation and analysis system (MINI HD, UVITEC, Cambridge).
3
Western Blot Analysis of ABCB1 in Cell Lines
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The cellular proteins were extracted from SW620, A549 and HepG2 cells with RIPA buffer containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA), and equal protein amounts (protein concentration of lysates measured by BCA assay, Thermo Fisher Scientific, Waltham, MA, USA) were subjected to SDS-PAGE. After electrophoresis proteins were transferred onto Trans Blot Turbo Mini PVDF membranes with Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA), blocked with 5% BSA and incubated with specific primary antibodies: mouse anti-human ABCB1 (clone F4, Sigma Aldrich, St. Louis, MO, USA). Specific HRP-conjugated secondary antibodies were used (Sigma Aldrich, St. Louis, MO, USA), and protein bands were detected using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) employing Mini HD (Uvitec, Cambridge, UK). MDR1/ABCB1 Sf9 insect membranes (Solvo Biotechnology, Budaörs, Hungary) were used as a positive control for the presence of ABC proteins, and β-actin serves as an internal control.
4
Western Blot Analysis of STAT Proteins
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Cells were lysed with 20 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Brij97, 2 mM Na Orthovanadate, and protease inhibitors (Roche Diagnostics, Complete Mini 04693124001). Proteins were resolved by 10% SDS-PAGE under reducing conditions, and Western blotting was carried out according to standard techniques, with rabbit anti-phospho-STAT1 (pY701) and anti-STAT1 anti-sera (Cell Signaling Technology, 9167 and 9172, respectively), mouse anti-phospho-STAT3 (pY705) and anti-STAT3 mAbs (BD Transduction Laboratories, 612,356 and 610,190, respectively) and α-tubulin mAb (Sigma-Aldrich, T6074).
Immunoreactive proteins were detected by ECL Prime (GE Healthcare, RPN2232) and a chemiluminescence gel documentation and analysis system (MINI HD, UVITEC, Cambridge, UK). Densitometric analyses of relevant bands and whole Western blot images are shown in theSupplementary Materials Figure S4 .
Immunoreactive proteins were detected by ECL Prime (GE Healthcare, RPN2232) and a chemiluminescence gel documentation and analysis system (MINI HD, UVITEC, Cambridge, UK). Densitometric analyses of relevant bands and whole Western blot images are shown in the
5
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using Complete Lysis buffer containing, proteases inhibitors (04719956001 Roche) and phosphatase inhibitors (04906845001 Roche). Preparation of nuclear extracts was performed by using NE-PER™ nuclear and cytoplasmic extraction reagents according to the manufacturer’s instructions. The Western blot of cell lysates was performed as previously described [40 (link)]. Antibodies: p-YAP rabbit mAb (#13008), YAP rabbit polyclonal (#4912), P-FAK rabbit (#2383), Cleaved caspase-3 rabbit mAb (#9664), cleaved PARP rabbit polyclonal (#9541), and P-AKT rabbit mAb (#4060) all purchased by Cell Signalling (MA, USA); and P-ERK mouse mAb (#sc-7383, Santa Cruz Biotechnology, USA), HDAC1 rabbit Ab (#H3284, Sigma-Aldrich), anti-β-tubulin (HRP) rabbit polyclonal (ab21058) and anti-GAPDH rabbit polyclonal antibodies (ab9385) from Abcam (Cambridge, MA, USA). Antibody binding was revealed by ECL Prime (RPN2232, GE Healthcare, Milan, Italy) and a chemiluminescence gel documentation and analysis system (MINI HD, UVITEC, Cambridge, UK).
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