Lysates were incubated at 95°C for 5 min. Denatured proteins were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was saturated in 5% milk PBST (5% dry powdered milk, 0.1% Tween-20, 1X PBS) for 1 hour and then incubated with primary antibodies overnight. Secondary antibodies (peroxidase-conjugated affinipure or IRDye secondary antibodies). Membranes were then developed with an ECL reagent kit (BioRad) or Li-COR imaging system. The western blots were quantified using Fiji software.
Ecl reagent kit
Manufactured by Bio-Rad
Sourced in United States, China
The ECL reagent kit is a laboratory equipment product designed for the detection of proteins in Western blot analysis. The kit contains solutions that facilitate the chemiluminescent detection of target proteins that have been separated by gel electrophoresis and transferred to a membrane.
Automatically generated - may contain errors
6 protocols using ecl reagent kit
1
Western Blot Protein Detection
2
Immunoblot Analysis of Hepatic Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblot analysis was carried out as described previously [37 (link)]. Hepatic tissues were lysed, proteins were extracted, and equivalent amounts of proteins (0.02–0.05 mg) from control and treatment groups were loaded into 10–12% of SDS-PAGE for electrophoresis, followed by transfer to PVDF membranes. The membranes were then blocked with 5% milk (non-fat dried) for 60 min and incubated with primary antibodies, such as Bax, Bcl-2, cleaved caspase-3, p65-NFkB, SOD, pAKT, tAkt, P-p38, tp38, and GAPDH, overnight at 4 °C. Membranes were washed and incubated with HRP conjugated specific secondary antibodies for 60 min at room temperature. Proteins were visualized on the membrane using an ECL reagent kit, and Western blot images were captured and analyzed using an instrument for gel imaging (Hercules, CA, Bio-Rad-USA).
3
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was carried out as described by Alasmari et al. [41 (link)]. Briefly, isolated proteins (30–50 μg) were electrophoresed on SDS-PAGE gels and then transferred onto PVDF membranes. After blocking with 5% nonfat dry milk for 60 min, blots were probed overnight at 4 °C with selective primary antibodies (against Ho-1, Nrf2, TNF-α, IL6, NF-κB-p65, cleaved-caspase-3, Bcl-2, Bax, and GAPDH, dilution 1:1000). The membranes were subsequently washed, then incubated for 60 min at room temperature with suitable HRP-conjugated secondary antibodies (dilution: 1:5000). The ECL reagent kit and gel imaging equipment (Bio-Rad, Hercules, CA, USA) were used to detect the presence of proteins on the membrane.
4
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In brief, protein lysates were separated by SDS-PAGE and were transferred to polyvinylidene fluoride (PVDF) blots. The blots were blocked (in 7.5% milk PBST solution) and were incubated with indicated primary and secondary antibodies. To visualize the targeted protein bands, the chemiluminescence (ECL) reagent kit (Bio-Rad, Shanghai, China) was applied. Data quantification was through the ImageJ software (NIH). Supplementary Fig. S1 contained the uncropped blotting images of the study.
5
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As reported before [42 (link), 43 (link)], total cellular protein lysates (40 μg proteins per treatment into each lane) were separated by 10-12% SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, St. Louis, MO, USA). The membranes were blocked and immuno-blotted with indicated primary and secondary antibodies. An enhanced chemiluminescence (ECL) reagent kit (Bio-Rad, Shanghai, China) was utilized to visualize interested protein bands. The ImageJ software (NIH) was utilized for data quantification.
6
OMV Peptide Detection via Dot Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As previously described,22 (link) the PVDF membrane was cut into a suitable size and marked, and then the membrane was activated in methanol for 5 minutes and soaked in transfer buffer. The membrane was dried on filter paper for 5 min at room temperature to eliminate residual moisture. Five microliters of OMV sample was dropped directly on the membrane, and the membrane was dried at 37°C for 5 minutes to fix the sample completely. The membranes were first blocked with 5% skim milk powder for 45 min, after which the peptides expressed in the OMV samples were tagged using mouse anti-HPV16 E7 antibody serum and horseradish peroxidase (HRP)-labeled anti-mouse IgG (H+L) (Invitrogen) as the primary antibody (1:500 dilution) and secondary antibody (1:10,000 dilution), respectively. The positive dots were developed using an enhanced chemiluminescence (ECL) reagent kit (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!