To determine if prostates from either the PC1 or PC2 group express the SV40 T antigen or the NE marker Synaptophysin, five prostates weighing approximately 0.1 g were randomly chosen at necropsy from mice aged 30 weeks for formalin fixation and paraffin embedding. For comparison, a prostate weighing over 4.5 grams from the PC1 group was also collected for immunohistological analysis. Tissue blocks were dissected at 5µm, adhered to a frosted glass slide and immersed in Clear-Rite™ (Thermo Fischer Scientific) for 2 changes of 10 minutes each. Tissue sections were rehydrated in gradient alcohols and endogenous enzyme activity was quenched using 0.3% H2O2 at RT and rinsed in TBST, followed by 1 hour of protein blocking using Superblock T20 Blocking Buffer (Thermo Scientific, cat#37356) at room temperature (RT). Primary antibodies mouse anti-SV40 LargeT-antigen (TAg) (BD Pharmingen, cat#61095) and mouse anti-synaptophysin (Thermo Fischer cat# MA5-16402), were diluted at 1:400 and 1:1500, respectively, in Superblock T20 Blocking Buffer for incubation overnight at 4°C. The next day, slides were washed in TBST for 3 × 2 minutes each followed by 1 hour incubation using EnVision™ + Dual link labelled HRP polymer (Dako, cat#K4065) at RT. Staining was visualized using DAB chromagen in DAB substrate buffer (Dako, cat#K4065) for 1 minute and counterstained with Haematoxylin for 2 minutes.
Superblock t20 blocking buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperBlock T20 blocking buffer is a ready-to-use solution designed to reduce non-specific binding in immunoassays and other protein-based applications. It contains a proprietary blend of proteins and surfactants to effectively block non-specific binding sites, thereby improving signal-to-noise ratios and assay performance.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti p53, by Cell Signaling Technology (2 mentions)
Reducing sample buffer, by Thermo Fisher Scientific (2 mentions)
Anti setdb1, by Santa Cruz Biotechnology (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Las 4000 mini, by Fujifilm (2 mentions)
Bis tris gel, by Bio-Rad (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Radioimmunoprecipitation assay ripa buffer, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Genepix 4000b microarray scanner, by Molecular Devices (1 mentions)
21 protocols using superblock t20 blocking buffer
1
Prostate Cancer Immunohistochemistry
2
Western Blot Analysis of AAV Proteins
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Transfected cells (48 h after transfection) were lysed with Radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO) and protease inhibitors (Roche, Basel, Switzerland). The cell lysates were prepared with loading buffer containing the reducing agents β-mercaptoethanol and then separated on a 4%–12% Bis-Tris gel (BioRad, Hercules, CA). Electrophoresed proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using the Turbo-blot Turbo system (BioRad, Hercules, CA). The membrane was blocked using SuperBlock T20 blocking buffer (Thermo Fisher Scientific, Waltham, MA) before probing with α-AAV2-Rep, 303.9 (Progen, Heidelberg, Germany) and horseradish peroxidase-conjugated secondary antibodies (Sigma-Aldrich, St. Louis, MO). Bound antibodies were detected with the enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific, Waltham, MA) and imaged using a Chemidoc imager (BioRad, Hercules, CA). AAV VP protein composition was determined by equal-volume loading of denatured AVB purified AAV particles, which were electrophoresed using Mini-protean Stain-free 4%–12% Bis–Tris polyacrylamide gels (BioRad, Hercules, CA). Gels were imaged using the Chemidoc system and analyzed using Image Lab software (BioRad, Hercules, CA).
3
Identifying LNC942 Binding Proteins
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The HuProtTM human proteome microarray (Wayen Biotechnologies, Shanghai) was used to identify potent LNC942 binding proteins.20 Briefly, proteome microarrays were blocked with a pre‐cold SuperBlock T20 Blocking Buffer (Thermo Scientific, USA) for 2 h with gentle agitation. The microarrays were washed with TBST (Tis buffered saline + Tween) and incubated with biotin‐labelled anti‐sense (control) or sense LNC942 probes for 1 h at RT. After being washed in TBST, the dried microarrays were scanned with a GenePix 4000B microarray scanner (Axon Instruments, USA) to evaluate the results. Both probes corresponding to a protein were required to have a z‐score of ⩾3 in two repeated array experiments was considered a positive interaction, resulting in 209 positive hits.
4
Western Blot Quantification and Analysis
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Lysates were heated to 95°C and supernatant was run on a 4–12% NuPAGE bis-tris precast gel in a NOVEX Xcell SureLock Mini-Cell System (Invitrogen, Carlsbad, CA) and transferred to a PVDF membrane (BioRad, Hercules, CA). The membrane was blocked using SuperBlock T20 Blocking Buffer (Thermo Scientific). Primary antibodies were applied and incubated overnight at 4°C (Table 1
5
Apolipoprotein E4 Detection by ELISA
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For the specific detection of apoE4 by ELISA, we used 96-well plates (Nunc Maxisorb flat-bottom 96 well plate) blocked with a BSA-based blocking solution (0.25% BSA solution in 15 mM borate buffer containing 0.05% polysorbate 20 and 100 mM NaCl, pH 8.5). Alternatively, Superblock T20 blocking buffer from ThermoFisher Scientific has been used in certain experimental setups at room temperature (RT) for 16 hours. After blocking, the plates were washed four times with TBS 0.1% polysorbate 20 (TBS-T), and then incubated with diluted plasma 1:200 in TBS-T for 1 hour at RT. After washing, mouse monoclonal antibody specific for apoE4 (clone 4E4, Novus Biologicals, #NBP1-49529) diluted 1:2,000 in TBS-T plus 20% blocking solution, was added onto the wells and incubated for 45 minutes at RT. Then, the plates were washed and anti-mouse IgG-peroxidase (1:10,000 in TBS-T plus 20% blocking solution, USBiologicals, IgG, H&L (X-Adsorbed HRP #I1904-06) was added for 30 minutes at RT. After washing, in order to visualize the antibody binding, TMB chromogenic substrate (BioRad, #1721066) was used for 15 minutes at RT and measured, after stop with 2.15 N sulfuric acid, using a spectrophotometer at 450 nm with reference at 750 nm.
6
Materials for Bioanalytical Assay Examples
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Example 1
The following example describes materials used in Examples 2-10. Optical fiber bundles were purchased from Schott North America (Southbridge, Mass.). Non-reinforced gloss silicone sheeting was obtained from Specialty Manufacturing (Saginaw, Mich.). Hydrochloric acid, anhydrous ethanol, and molecular biology grade Tween-20 were purchased from Sigma-Aldrich (Saint Louis, Mo.). 2.7-μm-diameter carboxyl-terminated magnetic beads were purchased from Varian, Inc. (Lake Forest, Calif.). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), and SuperBlock® T-20 Blocking Buffer were purchased from Thermo Scientific (Rockford, Ill.). Streptavidin-β-galactosidase (SβG) was purchased from Invitrogen, Sigma-Aldrich, or conjugated in house using standard protocols. Resorufin-3-D-galactopyranoside (RGP) was purchased from Invitrogen (Carlsbad, Calif.). The fiber polisher and polishing consumables were purchased from Allied High Tech Products (Rancho Dominguez, Calif.). Monoclonal capture antibody to PSA, monoclonal detection antibody to PSA, and purified PSA were purchased from BiosPacific. The Chromalink™ biotinylation reagent was purchased from Solulink, Inc (San Diego, Calif.). Purified DNA was purchased from Integrated DNA Technologies.
7
Western Blot Analysis of Colonic Tissues
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Colonic tissues and HT-29 cells were lysed with SDS lysis buffer, centrifuged, and then homogenized by Pierce BCA protein assay kit (Thermo Fisher Scientific). The protein samples were boiled for 10 min, electrophoresed in 10% SDS-PAGE, and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The blots were blocked with SuperBlock™ T20 blocking buffer (Thermo Fisher Scientific) and incubated at 4 °C with the appropriately diluted primary antibodies overnight. The signals were developed using the under ChemiDoc™ MP Imaging System (Bio-Rad).
8
Western Blot Assay for RNA Polymerase
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Cells were diluted 1,000-fold from overnight cultures and grown to an OD600 of 0.2 in 2 ml of medium before the addition of 80 µg ml–1 Rif for 1 h. Cells were then collected, resuspended in lysis buffer (10 mM Tris-HCl pH 7.5, 4% SDS), mixed with 4× lithium dodecyl sulfate (LDS) sample buffer (Invitrogen), and boiled at 95 °C for 10 min before loading on a NuPAGE 4–12% Bis-Tris precast gel (Thermo) for electrophoresis at 200 V for 40 min. Gel was then semidry transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) and incubated with the specified antibodies at 4 °C overnight in SuperBlock T20 blocking buffer (Thermo) before development the following day. Antibodies used were E. coli RNAP beta Monoclonal Antibody clone 8RB13 mouse mAB at 1:2,000 (663905; Biolegend) and LexA Antibody (E-7) at 1:200 (sc-365999; Santa Cruz). Gel intensity was quantified using GelQuant.NET.
9
Immunocytochemical Analysis of Stress Signaling Proteins
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Phospho-p38, cleaved ROCK1, and phospho-ATF2 were detected in IEC6 cells by immunocytochemistry. Cells grown on cover-slips in serum-free media (Dulbecco’s modified Eagle’s media) were treated with atRA 10 μM × 2h, washed, and then fixed with ice-cold methanol and acetone (1:1) × 10 min at -20°C, blocked (Superblock T20 blocking buffer, Thermo Scientific) × 30 min, and then incubated overnight at 4°C with primary antibody: mouse anti-phospho P38 (Tyr 182), mouse anti- ROCK1, or rabbit anti-phospho ATF2 (Thr 71) (all from Santa Cruz). Secondary staining was performed with Alexa Fluor 546-conjugated goat anti-mouse IgG and/or Alexa Fluor 488 conjugated chicken anti-rabbit IgG (Invitrogen) × 1h at room temperature. Nuclear staining was obtained with DAPI (Invitrogen). Fluorescence imaging was performed using a Zeiss LSM 710 confocal microscope.
10
Analyzing Cellular Signaling Proteins
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Western blot was performed as described previously. Samples were collected directly in 1X NuPAGE LDS sample buffer with 1X sample-reducing buffer (Invitrogen) and denatured at 95°C for 5 min followed by a centrifugation at 13200 rpm for 5 min. The supernatant was electrophoresed on a 4–12% Tris-HCl gel and transferred to nitrocellulose membranes (Invitrogen). After blocking with Superblock T20 blocking buffer (Thermo Scientific), the membranes were incubated with a primary antibody overnight at 4°C and then with a secondary antibody conjugated with alkaline phosphatase for 1 hour each at room temperature; the signal was detected using a chemiluminescence method. The following primary antibodies were used: anti-p53 (Cell Signaling, 1:1000); anti-phospho-p53 (S15; Cell Signaling, 1:1000); anti-GAPDH (Santa Cruz, 1:10000); anti-ERCC1 (Cell Signaling, 1:1000); and anti-PARP (Cell Signaling, 1:1000).
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