A total of 25 µg of crude venom of each individual was separated on a non-reduced 4–20% Tricine Gel for 90 min at a voltage of 125 using an XCell SureLock® Mini-Cell system(Invitrogen). Current was moderated using a Bio-Rad PowerPack power supply. Protein was transferred onto a 0.2 µm nitrocellulose membrane (Millipore) using a Trans Blot SD system (Bio-Rad) at 100 mA for 1 h and allowed to set overnight. Duplicate gels under the same conditions were stained with Simply Blue (Invitrogen) for 1 h. The membrane was then blocked with 5% BSA in TBST for 1 h, washed with PBS and incubated with the anti-r-mojastin 1 antibody (1:1000 dilution) overnight at room temperature. The membrane was washed three times with TBS and incubated with a biotinylated goat anti-rabbit antibody (1:30,000 dilution) for 1 h at room temperature. The membrane was washed three times with 0.05% Tween 20 in PBS and incubated with ExtrAvidin-peroxidase (SIGMA, USA) diluted to 1:2000 in PBS for 1 h at room temperature. After washing off the unbound avidin with TBST, the antigen-bound antibody was visualized with SigmaFAST™ 3,3′-diaminobenzidine tablets. Bands of interest were transferred to nitrocellulose using the same conditions, as previously described, excised, and sent for N-terminal sequencing at the Iowa State Sequencing Facility.
Power pack
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
The Power Pack is a versatile laboratory device designed to provide a stable and controlled power supply for various electrophoresis techniques. It is used to power electrophoresis equipment, such as gel electrophoresis rigs, ensuring consistent and reliable performance during experiments.
Automatically generated - may contain errors
Lab products found in correlation
Simplyblue, by Thermo Fisher Scientific (1 mentions)
Xcell surelock mini cell system, by Thermo Fisher Scientific (1 mentions)
Extravidin peroxidase, by Merck Group (1 mentions)
0.2 m nitrocellulose membrane, by Merck Group (1 mentions)
Trans blot sd system, by Bio-Rad (1 mentions)
Pierce bca kit, by Thermo Fisher Scientific (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Loading dye, by Bio-Rad (1 mentions)
1 kb dna ladder, by Promega (1 mentions)
Hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Mouse anti iκbα, by Cell Signaling Technology (1 mentions)
Rabbit anti ampk, by Cell Signaling Technology (1 mentions)
Westernbright quantum hrp substrate, by Advansta (1 mentions)
Rabbit anti p ampk t172, by Cell Signaling Technology (1 mentions)
Rabbit anti p iκbα ser32, by Cell Signaling Technology (1 mentions)
Rabbit anti adipor1, by Abcam (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
7 protocols using power pack
1
Venom Protein Separation and Identification
2
Western Blot Analysis of NK92 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NK92 cells were cultured with drug as described in legend. Cells were collected in radioimmunoprecipitation assay buffer (RIPA) (0.05% sodium deoxycholate, 150 nM NaCl, 50 nM Tris HCL, 0.1% SDS, 1% NP-40). Protein content was analyzed by Pierce BCA kit (Thermo 23225). 50 µg of protein was diluted in 1× loading buffer (SDS, bromophenol blue, 47% glycerol, Tris 0.5 M pH 6.8, 0.2 mM DTT-G) and heated to 90°C for 3 min. Prepared lysate was loaded into gradient gels (Bio-Rad 4561093) and run at 85V for 1.5 hour in Bio-Rad casing with a Bio-Rad powerpack in 1× running buffer (Tris, Glycine, SDS). Gels were transferred on nitrocellulose and run at 100V on bench for 1 hour in 1× transfer buffer (Tris, Glycine). Membranes were blocked in 5% BSA in TBS for 1 hour, washed, and incubated in primary diluted in 2% BSA in TBS overnight. Membranes were washed and incubated in Licor secondary (Licor 926–32211 and 925–68070) in 5% BSA in TBS for 1 hour. Membranes were washed and imaged on a Licor Odyssey Clx. Images were analyzed in Image Studio Lite V.5.2.
3
DNA Integrity Analysis via Agarose Gel
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Agarose gel electrophoresis was run to investigate the DNA integrity of each sample E. coli. In brief, the purified DNA (10μL) was mixed with loading dye (Bioline) and the sample loaded with on 1% agarose gel (containing ethidium bromide, 1 μl/mL) and subjected to electrophoresis using Mini slab, submerged electrophoresis apparatus and power pack (BioRad Laboratories, Richmond, CA), and run for 45 min at constant voltage of 100 volts. A 1kb DNA Ladder (Promega Corporation, Madison, WI) was used for molecular marking in a parallel well. The gel was viewed under UV translluminator and photographs obtained using the attached Gel Documentation System and software (Denville Scientific, Denville, PA).
4
Adiponectin Receptor and AMPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell homogenates (20 μg/well) were loaded onto 10% SDS polyacrylamide gels in denaturing conditions at 80 mA for 90 min and transferred electrophoretically (100 mA/blot, 2 h; Power Pack; Bio-Rad Laboratories, Inc., USA) to polyvinylidene fluoride (PVDF) membrane. Immunoblotting was performed as described previously [48 (link)]. Nonspecific binding was blocked with 5% non-fat milk powder in Tris-buffered saline-Tween containing 0.1% Tween-20 (PBS-T) for 1 h. Primary antibodies including rabbit anti-AdipoR1 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-AdipoR2 (1:1000, Boster Biological Technology, USA), rabbit anti-AMPK (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-p-AMPKT172 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-α-Tubluin (1:5000, Cell Signaling Tech. Inc., USA), rabbit anti-p-NF-κB p65S536 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-NF-κB p65 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-p-IκBα (Ser32) (1:1000, Cell Signaling Tech. Inc., USA), mouse anti-IκBα (1:1000, Cell Signaling Tech. Inc., USA) antibody were incubated at 4 °C overnight, followed by HRP-conjugated secondary antibodies (goat anti-rabbit, 1:5000 or rabbit anti-mouse, 1:5000; Dako, Glostrup, Denmark) at RT for 1 h. The immunoblot signals were visualized by Westernbright Quantum HRP substrate (advansta, USA).
5
Canine Genetic Marker Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cDNA was amplified and cross-checked with the marker in PCR in a sequential manner. PCR reaction mixtures were prepared for all canine experiments using the PCR kit (Invitrogen, USA) (Table-4 ).
The following protocol was found to be optimal for the reactions. An initial denaturation step of 95°C for 5 min was followed by 35 cycles of denaturation at 95°C for 1 min, annealing at primer-specific temperatures for 1 min, and extension at 72°C for 1 min. The program was completed with a final extension step of 72°C for 10 min. The samples were stored at 4°C until analysis and negative controls of RNA and water were run alongside to confirm the absence of genomic DNA contamination.
PCR products were run on a 2% agarose gel and 2% ethidium bromide. The samples were run in electrophoresis tank connected to a power pack (Bio-Rad, UK) to create band separation and analyze with Molecular Imger® Chemi Doc™ XRS+Imaging System.
The following protocol was found to be optimal for the reactions. An initial denaturation step of 95°C for 5 min was followed by 35 cycles of denaturation at 95°C for 1 min, annealing at primer-specific temperatures for 1 min, and extension at 72°C for 1 min. The program was completed with a final extension step of 72°C for 10 min. The samples were stored at 4°C until analysis and negative controls of RNA and water were run alongside to confirm the absence of genomic DNA contamination.
PCR products were run on a 2% agarose gel and 2% ethidium bromide. The samples were run in electrophoresis tank connected to a power pack (Bio-Rad, UK) to create band separation and analyze with Molecular Imger® Chemi Doc™ XRS+Imaging System.
6
Agarose Gel Electrophoresis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One gram of the agarose powder was dissolved in 100 ml of 0.5 x TBE solution by boiling in a hot plate. It was then allowed to cool to approximately 60 0 C and 2 drops of ethidium bromide was then added. The mixture was mixed gently and a gel thickness of about 4-5 mm was obtained by pouring into a taped electrophoresis tray with the comb and allowed to solidify for 20 minutes. The rubber dam and the comb were removed carefully to avoid damaging the gel. The tray was placed in an electrophoresis tank (Bio -Rad Power-pack) and 0.5 x TBE gel buffer was poured the first well of all gels before loading the samples. Five microlitres (5 µl) of loading dye was mixed with 5 µl of the sample. The mixture was loaded into a comb well. Electrophoresis was then initiated at 60-100 V until the loading dye has migrated three quarter of the gel. Bands of the DNA on the agarose gel containing the extracted samples were observed through transillumination with the use of ultra-violet ray transilluminator (Fisher Biotech).
7
Protein Fractionation and 2D Gel Electrophoresis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For protein fractionation, 10 µL of extracted proteins were denatured by boiling for 5 min and loaded into gel wells after mixing with 10 µL of 4X concentrated sample loading buffer. Electrophoresis was carried out at 100 volts using a Bio-Rad power pack (Rad, 2012) . After reaching the bottom of the resolving gel, fractionated gels were removed and placed into a staining solution. For 2-D gel electrophoresis, fractionated proteins were separated according to their charge using Isoelectric Focussing (IEF) on the IPG Runner cassette system at pH 3-10. IEF was performed using an electric voltage of 175 to 2000 volts over 45 min and 2000 volts overnight. After the completion of IEF, the focused strips were run immediately on the Sodium-Dodecyl-Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. Protein fractionation was conducted as described by Laemmli (1970; (link)Rad, 2012) .
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!