Western blot analyses of protein samples were performed as described previously for rabbit anti-Tse1 (diluted 1:5,000; Genscript), rabbit anti-FLAG (diluted 1:5,000; Sigma), rabbit anti-VSV-G (diluted 1:5,000; Sigma), rabbit anti-Hcp1 (P. aeruginosa) (diluted 1:5,000, Genscript) and detected with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (diluted 1:5,000; Sigma) (Ahmad et al., 2019 (link)). Rabbit anti-Hcp (P. protegens) was used at a 1:5000 dilution. Western blots were developed using chemiluminescent substrate (Clarity Max, Bio-Rad) and imaged with the ChemiDoc Imaging System (Bio-Rad).
Clarity max
Manufactured by Bio-Rad
Sourced in United States
Clarity Max is a high-performance Western blotting imaging system designed for reliable and accurate detection of proteins. It features a sensitive CCD camera, a powerful imaging engine, and intuitive software for data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc imaging system, by Bio-Rad (5 mentions)
Rabbit anti vsv g, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Merck Group (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Chemidoc, by Bio-Rad (3 mentions)
Rabbit anti flag, by Merck Group (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Loading dye, by Bio-Rad (1 mentions)
Pierce radioimmunoprecipitation assay ripa buffer, by Thermo Fisher Scientific (1 mentions)
Pierce micro bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail 1, by Merck Group (1 mentions)
38 protocols using clarity max
1
Protein Expression Analysis via Western Blot
2
Protein Detection via Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analyses of protein samples were performed as described previously using rabbit anti-VSV-G (diluted 1:5,000; Sigma), rabbit anti-Hcp (diluted 1:5,000) and detected with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (diluted 1:5,000; Sigma)6 (link). Western blots were developed using chemiluminescent substrate (Clarity Max, Bio-Rad or SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermofisher) and imaged with the ChemiDoc Imaging System (Bio-Rad).
3
Western Blot Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were lysed on ice in Pierce radioimmunoprecipitation assay (RIPA) buffer (Thermo, 89900) containing complete EDTA-free protease inhibitor cocktail (Sigma/Roche, 11836170001) and debris was pelleted. Protein content was determined using Pierce Micro bicinchoninic acid (BCA) Protein Assay kit (Thermo, 23235). Samples were prepared in 4x loading dye (Bio-Rad Laboratories, Hercules, CA), boiled at 95°C for 5 min, and loaded in 4%-15% gradient Mini-Protean TGX Stain-Free precast gels (Bio-Rad). Gels were run at 175V and transferred to a 0.2-μm-pore-size nitrocellulose membrane by the semidry transfer method. Blots were blocked in 1% casein buffer/Tris-buffered saline (TBS) for 3 hours at 4°C. Primary antibodies were diluted in 1% casein and incubated overnight at 4°C. Blots were washed three times with TBS with 0.01% Tween 20 (TBST) and incubated with secondary antibody diluted in TBST for 1 hour at room temperature. Horseradish peroxidase secondary antibodies were diluted 1:10,000. Blots were washed three times with TBST and then incubated with ClarityMax according to manufacturer’s recommendation (Bio-Rad, 1075062) for HRP secondary antibodies. Blots were imaged on a ChemiDoc MP imaging system.
4
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in Laemmli buffer [(100 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% glycerol and 200 mM DTT (dithiothreitol)] and then processed for Western blot analysis, as previously described [33 (link),36 (link)]. Briefly, after SDS-PAGE, proteins were transferred on PVDF membrane (Merck-Millipore, Burlington, MS, USA) which was then blocked with 5% milk in PBS for 30 min and then incubated with the appropriate primary antibodies (over-night at 4 °C) and secondary HRP-conjugated antibodies (1 h at room temperature), all prepared in 1% milk in PBS. Chemiluminescence reaction occurred using Clarity or Clarity Max (Biorad, Hercules, CA, USA) and the signal was captured by ChemiDoc MP Imaging Systems (Biorad). Densitometric analysis was performed using Image Lab software (Biorad).
5
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA (Radioimmunoprecipitation Assay) buffer (50mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) plus protease inhibitor cocktail (Roche, Mannheim Germany) and phosphatase inhibitors (phosphatase inhibitor cocktail 1 from Sigma-Aldrich). Lysates were loaded onto SDS-PAGE and separated proteins were transferred onto polyvinylidene fluoride (PVDF) membrane from Millipore (Billerica, MA, USA) and probed with the primary antibody diluted in 2% milk in PBS (Phosphate-Buffered Saline) followed by HRP-conjugated secondary antibody, as previously described [23 (link)]. Bands were visualized using Western blot Luminol Reagent (Santa Cruz), WesternBright ECL kit (Advansta, Menlo Park, CA, USA), Clarity (BioRad, Hercules, CA, USA), or Clarity Max (BioRad, Hercules, CA, USA), and signals were captured on a film or using Bio-Rad ChemiDoc MP Imaging Systems (Hercules, CA, USA).
6
Quantification of Histone Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared using RIPA buffer (50 mmol/L Tris–HCl, pH 7.4; 150 mmol/L NaCl; 1% Triton X-100; 1% sodium deoxycholate) consisting of protease and phosphatase inhibitors. Equal protein concentration was loaded for all samples, separated on 12–18% polyacrylamide gel, and transferred onto PVDF membrane of pore size 0.45 µm. The transfer was carried out applying 300 mA constant current for 4 h at 4 °C. The membrane was blocked in 5% BSA in TBST for 1 h and incubated with primary antibody overnight at 4 °C. Further, membrane was washed 3 times with TBST and then incubated with secondary antibody for 1 h at RT. The blots were washed 3 times with TBST and developed using a chemiluminescent substrate (ClarityMax, Biorad). Antibodies used in study are provided in Table 3 .
Antibody used in the study
| Protein | Company | Catalog number | Dilution | Purpose |
|---|---|---|---|---|
| H3 | Sigma | H-0164 | 1:5000 | WB |
| pAKT | Cell Signaling | 43966 | 1:1000 | WB |
| pH2AX | Millipore | 05-636 | 1:5000 | WB |
| H3K9ac | Millipore | 04-1003 | 1:5000 1:100 1:100 | WB IF IHC |
| H3K27ac | abcam | ab4729 | 1:5000 2 µg/10 µg chromatin | WB ChIP |
| H3K56ac | abcam | 76307 | 1:3000 | WB |
| H4K5ac | Millipore | 06-759 | 1:5000 | WB |
| H4K16ac | Millipore | 07-329 | 1:8000 2 µg/10 µg chromatin 1:100 | WB ChIP IF |
| H3K9Me3 | abcam | 8898 | 1:4000 | WB |
| Pan-acetyl lysin | abcam | ab80178 | 1:1000 | WB |
| Secondary anti-rabbit | Cell Signaling | 7074 | 1:8000 | WB |
| Secondary anti-mouse | Sigma | A-4416 | 1:5000 | WB |
| Caspase 3 | Cell signaling | 9662 | 1:100 | IHC |
| β-actin | Sigma | A-5316 | 1:10,000 | WB |
7
High-throughput SARS-CoV-2 Antibody Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum samples were probed using 1:50 dilution and DBS samples using a 1:20 dilution on the pillar plate and reacted for 15 minutes at room temperature. The plate was then washed with Tris-buffered saline with Tween 20 (TBST) (Amresco) buffer 3 x 5 minutes each. The plate was incubated with the secondary antibody (1:2000 dilution of Goat Anti-Human IgG HRP and Goat Anti-Human IgM HRP and Goat Anti-Human IgA HRP individually) for 15 minutes at room temperature. The plates were then washed with TBST buffer followed by washing with DI Water. The plates were finally dried completely before adding chemiluminescent substrate (Clarity Max from Bio-Rad) and scanned for five minutes on a standard Chemiluminescence Imager. For the Enhanced IgM Assay, the serum was pre reacted with Goat anti-human IgG Fc fragment prior to the remaining assay steps to increase the sensitivity of IgM and IgA detection. Assaying is performed on automated liquid handlers enabling a throughput of 100,000 samples per day.
8
Protein Detection via Western Blot
9
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from iPS-CM by homogenization in RIPA buffer (Sigma Aldrich) containing protease inhibitor cocktail (Roche). For each sample, 10 µg of proteins were separated in reducing conditions by 4–12% SDS-PAGE and transferred to PVDF membrane (ThermoFisher Scientific). The membrane was blocked for 2 h at room temperature with 5% powdered skim milk in Tris-buffered saline pH 7.4 with 0.1% Tween (T-TBS). The membrane was then incubated with Apoptosis Western Blot Cocktail (#ab136812, Abcam, 1:250), or HSP90 (#610418, BD Biosciences, 1:3000). Antibodies were diluted in T-TBS with 3% bovine serum albumin (BSA, Sigma) and incubated overnight at 4 °C. Blots were washed five times with T-TBS and incubated with HRP-conjugated secondary antibodies anti-mouse (#12/2013, Cell Signaling Technology, Danvers, MA, USA, 1:10000) and anti-rabbit (#07/2014, Cell Signaling Technology, 1:10000) in T-TBS with 5% powered skim milk for 1 h at room temperature. Blots were washed five times with T-TBS. Bound antibodies were detected through enhanced chemiluminescence (Clarity Max, Bio-Rad, Hercules, CA, USA). Bands were analyzed by densitometry, using Fiji-ImageJ software.
10
Subcellular Fractionation and Immunoblotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Subcellular fractionation was performed using an established method (69 (link)). Briefly, parasites were under a freeze-thaw process followed by centrifugation. The supernatant was harvested as the cytoplasmic fraction. The pellet was then suspended in RIPA Buffer followed by centrifugation, and the supernatant was used as the nuclear fraction. Goat anti-GFP antibodies (1:2,500, ab6673, Abcam, RRID:AB_305643, USA), mouse monoclonal anti-γ-H2A.X (1:2,500; clone JBW301, Sigma-Aldrich, AB_2924829, USA), and hATM [1:1,000, 2C1(1A1), ab78, Abcam, RRID:AB_306089], rabbit anti-histone H3 antibodies (1:1,000 dilution; Millipore), and rabbit anti-Plasmodium aldolase antibodies (1 µg/mL, ab207494, Abcam, USA) were used as primary antibodies. HRP-conjugated goat anti-rabbit IgG (1:5,000, Millipore) or rabbit anti-goat or mouse IgG (ab6741, RRID:AB_955424, ab6728, RRID:AB_955440, Abcam, USA) was used as the secondary antibodies. The results were visualized with the ECL detection system (Clarity Max, 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!