For Western blot, proteins were blotted on nitrocellulose membranes using a Trans‐Blot Turbo™ Transfer System (Bio‐Rad). Membranes were blocked with 5% nonfat dry milk in TBST buffer for 1 h. Primary antibodies were incubated for 1 h at room temperature or overnight at 4°C with shaking. Secondary HRP‐conjugated antibodies were diluted 1:10,000 in TBST and incubated for 1 h at room temperature. Blots were developed using Radiance ECL substrate (Azure Biosystems) or SuperSignal™ West Femto (Thermo Fisher Scientific) or Clarity western ECL (Bio‐Rad) substrates on an Amersham Imager 680 or Bio‐Rad ChemiDoc. Automated capillary electrophoresis immuno‐quantification runs were conducted on a WES instrument (ProteinSimple) according to manufacturer recommendations. The indicated MW are determined based on an internal standard of known MW spiked in to every capillary.
Amersham imager 680
Manufactured by Bio-Rad
Sourced in United States
The Amersham Imager 680 is a compact and versatile imaging system designed for a range of life science applications. It utilizes a high-resolution CCD camera to capture images of gel- and blot-based assays, including Western blots, Northern blots, and chemiluminescent or fluorescent samples. The system provides a flexible and user-friendly interface for image acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Radiance ecl substrate, by Azure Biosystems (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Goat anti rabbit, by Merck Group (1 mentions)
Goat anti mouse, by Abcam (1 mentions)
Anti hur, by Santa Cruz Biotechnology (1 mentions)
Anti cxcl10, by Abcam (1 mentions)
Western ecl substrate, by Bio-Rad (1 mentions)
Anti hsp90, by Abcam (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Epiquik total histone extraction htkit, by Epigentek (1 mentions)
Anti h3k9me2, by Epigentek (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Cocktail 2, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using amersham imager 680
1
Western Blot Analysis and Quantification
2
Western Blot Analysis of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pf-iRBC were saponized using 0.2% saponin in PBS (S7900-Sigma) and then were lysed using RIPA buffer. EVs at a concentration of 1.5–2 × 1011 particles per ml were lysed in 20 µl of 5X RIPA buffer were sonicated in a bath sonicator for 10 s. THP-1 cells were lysed using RIPA buffer. Then, 120 μg of the obtained protein was subjected to gel electrophoresis; CXCL10 WB using 4–20% Bis-Tris gradient gels (GeneScript) and transferred into PVDF membranes (Bio-Rad); the rest of WB using 10% SDS-TRIS gels transferred into nitrocellulose membranes (Bio-Rad). The membrane was blocked for 1 hour with 5% skim milk in PBS-Tween 20 0.05%. The following primary antibodies were used in this work: anti-CXCL10 produced in rabbit (Abcam) for 2 h or anti-HSP90 produced in mouse (Abcam) for 1 hour, both diluted 1:1000; Anti-HUR produced in rabbit (Santa Cruz), anti-GAPDH produced in rabbit (Abcam), and anti-Dematin produced in rabbit (Abcam) for 1 h, all diluted 1:1000. The secondary antibodies used were goat anti-rabbit (Sigma-Aldrich) diluted 1:20,000 and goat anti-mouse (Abcam) diluted 1:2000, both conjugated with HRP and incubated for 40 minutes. The membrane was developed using Western ECL Substrate (Bio-Rad) and captured by Amersham imager 680 v2.0.0.
3
Quantifying H3 K9me2 Levels in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the ratio of H3 K9me2/H3 total N2 (WT) and CL2006 animals were incubated in liquid culture with vehicle (DMSO 1 %), or 0.1 μM of G9a inhibitor in a 96‐well plate format as already mentioned. For chronic treatment, four‐day‐old animals were collected with M9 buffer. Histone extraction was performed following the manufacturer's instructions (EpiQuik Total Histone Extraction HT Kit, EpiGentek, #OP‐0007‐192). The samples were resolved in a 14 % SDS‐gel, as previously described.[12] To capture chemiluminescence signals were used Amersham Imager 680 and Western blot quantifications were performed using ImageLab software (Bio‐Rad). Immunoblots were probed with anti‐H3 K9me2 (1 : 1000) (Epigentek, #A‐4035), and anti‐H3 total (1 : 1000) (Cell signaling, #9715).
4
Western Blot Analysis of Mouse Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For WB, brain tissues (n = 6 mice per group) were homogenized with lysis buffer containing phosphatase and protease inhibitors (Cocktail II, Sigma, St. Louis, MI, USA), and Bradford’s method was used to determine the protein concentration. Aliquots of 15 µg of protein were separated by SDS-PAGE (10–16% gels) and transferred into a PVDF membrane (Millipore, Burlington, MA). Afterwards, the membranes were blocked with 5% BSA for 1 h at room temperature. The blockage was followed by overnight incubation at 4 °C with the primary antibodies (Table S2 ). The next day, the membranes were washed and incubated with secondary anti-mouse or anti-rabbit antibody for 1 h at room temperature (Table S2 ). The proteins were revealed using chemiluminescence-based detection kits (Thermofisher and ECL kit, Millipore, Burlington, MA, USA) and images were obtained using an Amersham Imager 680 (BioRad, Hercules, CA, USA). Then, quantitative analysis was carried out using ImageLab Software (Bioke, CA, USA) and the results were expressed in arbitrary units (AU), considering the control group as 100%. To ensure that the differences between the samples were not a result of inaccurate sample preparation, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a protein charge control.
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!