Cells were washed twice with ice-cold PBS and harvested in cold sonication buffer [150 mM NaCl, 50 mM Tris (pH 8), 1% NP-40, 0.5% deoxycholate, 25 mM MG132, and 2X protease inhibitor mixture (Roche, cOmplete EDTA-free)]. Cells were sonicated (QSonica Sonicator system fitted with a 1.6-mm tip) and centrifuged at 9391 RCF for 5 min at 4°C. Lysates were incubated overnight at 4°C with specific antibodies or normal IgG (Millipore, Billerica, MA). Lysates were then incubated with protein A/G beads (Santa Cruz Biotechnology) for 3 h, followed by 4 washes with ice cold wash buffer [50 mM NaCl, 20 mM Tris (pH 8.3), 0.5% Sodium deoxycholate, 0.5% Nonidet P-40, 2 mM EDTA, 25 μM MG132, and 2X protease inhibitor mixture]. Immunoprecipitated proteins were resolved by SDS/PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). Blots were incubated with different primary antibodies followed by incubation with HRP-conjugated secondary antibodies. For CBFβ IP, Clean-Blot IP Detection Reagent was used (Cat. #21230, Thermo Scientific, Rockford, IL). Chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA) was visualized using the BioRad ChemiDoc SRS device.
Chemidoc srs device
Manufactured by Bio-Rad
Sourced in Morocco, United Kingdom, United States
The ChemiDoc SRS device is a compact, multipurpose imaging system designed for a range of laboratory applications. It captures high-quality images of gel-based assays, blots, and other samples using sensitive CCD camera technology. The device provides consistent, reproducible imaging results, enabling researchers to document and analyze their experimental data effectively.
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5 protocols using chemidoc srs device
1
Immunoprecipitation and Western Blot Analysis
2
CHIKV Protein Expression Analysis
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The expression screening was carried out in Vero cells by transduction of ChAdOx1 sCHIKV, using a MOI = 10. Three days after transduction, the cells and supernatants were harvested, the supernatant was concentrated with a 100 kDa Amicon® spin column (Millipore UK, LTD). The 5x concentrated fraction, the non-concentrated fraction (input) and flow through fraction was also recovered. Cells and supernatants samples were boiled at 100 °C for five minutes in Laemli buffer. Equal amounts of total cell extract and cell supernatants were resolved by SDS/PAGE and transferred to PVDF membranes. Blots were blocked with 1X PBS-Tween- 5% milk and incubated with an anti-CHIKV E1 antibody (AZ 1253, Aalto BioReagents, Dublin, Ireland) at 1:500 dilution, and anti-CHIKV envelope seropositive mice sera (1:500), followed by incubation with HRP-conjugated secondary antibody (1:5000). Chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA, USA) was visualised using the ChemiDoc SRS device (BioRad, Watford H., UK).
3
ZIKV Envelope Protein Detection
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Cells were washed twice with ice-cold PBS and harvested in cold 1× PBS. 1× and 4× Laemmli buffer were added to cells and supernatants, respectively, and boiled at 100 °C for 5 min. Equal amounts of total cell extracts and 15 μl of cell supernatants were resolved by SDS/PAGE and transferred to polyvinylidene fluoride membranes. In some cases, supernatants were 10× concentrated using Amicon Ultra-0.5 ml centrifugal filters. Blots were blocked with 1× PBS-Tween-5% milk and incubated with anti-ZIKV envelope seropositive mice sera (1:500) or a mAb versus ZIKV envelope (AZ1176, Clone 0302156), Aalto Bio Reagents), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000). Chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA) was visualised using the BioRad ChemiDoc SRS device. Uncropped images of the blots are shown in Supplementary Figs. 2 , 5 , 9 , 10 and 11 ).
4
Immunoprecipitation and Western Blot Analysis
5
Immunoblotting for C-Tag and Plasmodium VK Repeats
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Supernatants from transfected HEK293 cells were boiled at 100 °C for 5 min in laemli buffer. Equal amounts cell supernatants were resolved by SDS/PAGE and transferred to PVDF membranes. Blots were blocked with 1× PBS-Tween- 5% milk and incubated with an anti-C-Tag antibody (CaptureSelect™ Biotin Anti-C-tag Conjugate at 1:2,000 dilution, supernatants from a MRA-185 hybridoma cell line (2E10.E9) that recognize the VK247 repeats, and supernatants from a MRA-184 hybridoma cell line (2F2) that recognize the VK210 repeats. followed by incubation with HRP-conjugated secondary antibody (1:5,000). Chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA) was visualized using the BioRad ChemiDoc SRS device.
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