PCa cells were seeded and exposed to TOPO at the estimated 72 h MTT IC50 for each treatment protocol (CONV and METRO). Post-treatment cells were lysed in cell lysis buffer (Thermo Scientific™ RIPA Buffer). Equal amount of protein loaded onto 4–15% Criterion™ TGX Stain-Free™ Precast Gels. Proteins will be separated under reducing conditions and then transferred to a PVDF membrane using a Trans-Blot Turbo Mini transfer pack from Bio-Rad (Hercules CA, United States ). Nonspecific binding was limited by incubating the membrane in blocking buffer (2.5% (w/v) casein, pH 7.6, 150 mM NaCl, 10 mM TRIS-HCl, and 0.02% sodium azide). Membranes were incubated with primary antibodies for target protein (1:1000) for overnight and then with the appropriate secondary antibody (1:10,000) at room temperature. Immunoreactivity was detected by Pierce ECL Western Blotting substrate (Bio-Rad, CA). Images were captured and quantify by Gel Doc™ EZ Gel Documentation System and ImageLab™ Software (Hercules CA, United States ). Densitometry analysis was performed using standard image analysis software ImageJ.
Trans blot turbo mini transfer pack
Manufactured by Bio-Rad
Sourced in United States
The Trans-Blot Turbo Mini Transfer Packs are a laboratory equipment product designed for protein transfer in western blotting applications. The packs contain the necessary components, such as transfer membranes and filter papers, to facilitate the efficient transfer of proteins from polyacrylamide gels to a solid support.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Mini protean tgx stain free protein gel, by Bio-Rad (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce ecl western blotting substrate, by Bio-Rad (1 mentions)
Ecl western blotting detection kit, by GE Healthcare (1 mentions)
Chemi genius 2 bio imaging system, by Syngene (1 mentions)
Pierce 660 nm protein assay kit, by Thermo Fisher Scientific (1 mentions)
Clarity max ecl, by Bio-Rad (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Tris glycine sds running buffer, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
9 protocols using trans blot turbo mini transfer pack
1
Western Blot Protein Analysis
2
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were transferred electrophoretically from SDS–PAGE gels to PVDF membranes using the Trans-Blot® Turbo™ Mini Transfer Pack (Bio-Rad, United States). The membranes were then blocked for 1 h at room temperature with 1% non-fat milk in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, [pH 7.4]), washed three times for 10 min with TPBS (PBS plus 0.1% Tween 20) and probed for 2 h at room temperature with the anti-long chain acyl-ACP thioesterase 3-2 antibody (1:5,000 in TPBS; Dörmann et al., 1995 (link)). The membrane was washed gently three times for 10 min with TPBS buffer and then antibody binding was detected with a goat anti-rabbit IgG peroxidase conjugated antibody (1:10,000 in TPBS: Thermo Scientific, United States) at room temperature for 1 h. The membrane was washed again three times with TPBS and the protein bands were then visualized using the ECL western blotting detection kit (GE Healthcare), following the manufacturer’s instructions. Images were recorded in a Chemi Genius2 Bio-Imaging System (Syngene, India).
3
Western Blot Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitor cocktail (Roche, Grenzach-Wyhlen, Germany or Thermo Fisher Scientific). Protein lysates were scraped against Eppendorf rack for 20 times and centrifuged with 15 000 rpm for 15 min at 4°C. The protein concentration of supernatant lysates was determined using the Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific) and a NanoDrop1000 Spectrophotometer (Thermo Fisher Scientific). Proteins were separated in a Mini-Protean TGX Stain-Free Precast 4–15% Gel (Bio-Rad) using Tris/Glycine/SDS running buffer (Bio-Rad). Proteins were transferred to a 0.2 μm polyvinylidene difluoride (PVDF) transfer membrane either using a Trans-Blot Turbo Mini Transfer Pack (Bio-Rad) in a Trans-Blot Turbo (Bio-Rad) or using a Mini Trans-Blot Cell (Bio-Rad) in a Mini-Protean Tetra Cell (Bio-Rad). Following antibody incubation, membranes were developed using Clarity Max ECL (Bio-Rad) and a ChemiDoc MP imaging system (Bio-Rad).
Antibodies and their working concentrations are listed inSupplementary Table S5 .
Antibodies and their working concentrations are listed in
4
Protein Quantification and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In total, 25 × 104 cells were treated with 50 µl of cell lysis buffer (Complete Lysis-M; Roche, Basel, Switzerland) containing protease inhibitors (Complete protease inhibitor cocktail; Roche) and phosphatase inhibitors (PhosSTOP; Roche) and frozen at –80°C until further use. Protein concentration was determined using a PierceTM BCA Protein Assay Kit (Thermo Fisher Rockford, IL, USA), and 5 µg of protein was co-incubated with Laemmli sample buffer containing 2-mercaptoethanol at 95°C for 5 min. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Trans-Blot Turbo Mini Transfer Packs; Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). The primary antibodies used were rabbit polyclonal anti-TOMM20 (ab56783; Abcam, Cambridge, UK) or anti-TFAM (ARP31400_P050; Aviva Systems Biology, San Diego, CA, USA). Horseradish peroxidase-conjugated donkey anti-rabbit antibodies (Abcam) were used as secondary antibodies. The membranes were digitized using the ImageQuant LAS 4000 Biomolecular imager and ImageQuant software (GE Healthcare, Buckinghamshire, UK). The expression level of each protein was normalized to that of β-actin. Western blotting was repeated four times.
5
Quantitative Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysates were prepared with 2% sodium dodecyl sulphate and protease inhibitor cocktail (P2714, Sigma-Aldrich, France). Protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific). Proteins (25 μg) were separated by denaturing SDS-PAGE (4–15% Mini-PROTEAN® TGX Stain-Free™ Protein Gels, Bio-Rad) and transferred onto polyvinylidene fluoride membranes (Trans-Blot Turbo Mini Transfer Packs, Bio-Rad). Dilution (in 1% nonfat dry milk, 1X TBS 0.1% Tween-20), incubation conditions, and reference of primary antibodies used are described in Supplemental Table S5 . Antibody binding was revealed with an anti-rabbit (1:10,000; Jackson Immuno-Research Laboratories) IgG coupled to horseradish peroxidase, using ECL chemiluminescence kit (Clarity Western ECL substrate, Bio-Rad) and read on ChemiDoc Imaging System (Bio-Rad). Band quantification was performed on Image Lab software (Bio-Rad) using total protein stain as normalization.
6
Western Blot Protocol for Cell Lysates
7
HDAC4 Protein Extraction and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
20–30 mg of frozen tissues were homogenated in 200 μl modified lysis buffer (25 mM Tris-HCl pH 7.6, 2 mM EDTA pH 8, 250 mM Sucrose, 0.1% TritonX-100, 5 mM DTT, 1 mM PMSF, 200 mM SOV4, 10 mM NaF, protease inhibitor cocktail 1X; Sigma-Aldrich). A standard RIPA buffer (25 mM Tris-HCl, 1% NP-40, 1% Na-deoxycholate, 150 mM NaCl, 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4, protease inhibitor cocktail 1X; Sigma-Aldrich) was instead used for the extraction of total proteins from cell cultures. Protein samples were quantified by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and stored at −20 °C.
20 µg of lysate samples were separated on Mini-PROTEAN TGX Stain-Free Gels (4–20%, Bio-Rad). The gel was transferred to a nitrocellulose membrane (Bio-Rad) for 10 min using the Trans-Blot Turbo Transfer System (Bio-Rad) with Trans-Blot Turbo Mini Transfer Packs (Bio-Rad). The membrane was activated for 1 minute and then incubated with an antibody against HDAC4 (Cell Signaling, #2072) at a dilution of 1:500. Immune complexes were detected using the ChemiDoc Touch Imaging System (Bio-Rad)after incubation with Clarity Western ECL Substrate (Bio-Rad). HDAC4 protein expression was quantified and normalized to stain free gel loading using ImageLab software (version 5.2.1, Bio-Rad).
20 µg of lysate samples were separated on Mini-PROTEAN TGX Stain-Free Gels (4–20%, Bio-Rad). The gel was transferred to a nitrocellulose membrane (Bio-Rad) for 10 min using the Trans-Blot Turbo Transfer System (Bio-Rad) with Trans-Blot Turbo Mini Transfer Packs (Bio-Rad). The membrane was activated for 1 minute and then incubated with an antibody against HDAC4 (Cell Signaling, #2072) at a dilution of 1:500. Immune complexes were detected using the ChemiDoc Touch Imaging System (Bio-Rad)after incubation with Clarity Western ECL Substrate (Bio-Rad). HDAC4 protein expression was quantified and normalized to stain free gel loading using ImageLab software (version 5.2.1, Bio-Rad).
8
Protein Expression Analysis Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were obtained by lysing cells in TNN buffer supplemented with 1× Complete ULTRA protease inhibitor (Roche, Basel, Switzerland) and 1× PhosSTOP (ThermoFisher Scientific, Waltham, MA, USA). A total of 25 µg protein was separated on 4–10% Criterion TGX Stain-Free Protein Gels (BioRad, Hercules, CA, USA) or Mini-PROTEAN TGX Stain-Free Protein Gels (BioRad) and immediately transferred to Trans-Blot Turbo Midi PVDF Transfer Packs (BioRad) or Trans-Blot Turbo Mini Transfer Packs (BioRad). The blots were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at RT followed by incubation with anti-human CDKN2A antibody (Abcam, ab108349, 1:1000), anti-human Mesothelin antibody (Abcam, ab93620, 1:150), anti-human Podoplanin antibody (Abcam, ab236529, 1:500), and anti-human NF2 antibody (Abcam, ab109244, 1:10,000) overnight at 4 °C. The detection antibody HRP anti-rabbit IgG (BioLegend, San Diego, CA, USA, 410406, 1:1000) was incubated for 1 h at RT. Membranes were imaged using either Clarity Western ECL Substrate (Biorad) or SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) on a Fusion FX (Vilber Lourmat, Eberhardzell Germany). GAPDH and α-Tubulin were used as loading controls, anti-human GAPDH antibody (ThermoFisher Scientific, MA5-15738, 1:1000), and anti-human α-Tubulin antibody (Sigma-Aldrich, T5168, 1:1000).
9
Immunoblotting Protocol for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immunoblotting was performed as described previously [63] . Briefly, cell pellets were resuspended in a 50-150μL of cell lysis buffer [20mM Tris-HCl [pH 7.5], 150mM NaCl, 1mM Na 2 EDTA, 1mM EGTA, 1% [vol/vol] Triton X-100, 2.5mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na 3 VO 4 , and 1X complete EDTA-free protease inhibitor cocktail (Roche). Protein lysates were quantified with Bio-Rad protein assay and 0.5-2μg/μl solutions were prepared using 4X Laemmli sample buffer (Bio-Rad). 10-30μg of samples were loaded into 4-15% mini-protean TGX precast gels (Bio-Rad) and transferred to 0.2μm nitrocellulose membrane with Trans-blot turbo mini transfer packs (Bio-Rad). After blocking in 5% milk in PBS for 60 minutes, membranes were incubated with the primary antibody for 16 hours at 4ºC, followed by incubation with a secondary antibody for 60 minutes at room temperature. Antibodies were diluted in 5% bovine serum albumin in PBS + 0.1% tween20. The membranes were imaged using Chemidoc imaging system (Bio-Rad).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!