Total protein lysates and subcellular fractions were prepared from muscle homogenates and primary fibroblasts from the index patient and two controls. Protein samples were separated on NuPAGE™ Novex™ 4–12% Bis-Tris Protein Gels. Following electrophoresis, the separated proteins were transferred onto PVDF membrane using the iBlot® 2 Dry Blotting System (Thermo Fisher Scientific, UK) according to manufacturer’s instructions. The membrane was then probed for the following proteins: Total OXPHOS Rodent WB antibody cocktail (Abcam, ab110413), Anti-DRP1 (ab56788), Anti-SMCR7L (MID51) (ab89944), Anti-OPA1 (ab119685), Anti-Mitofusin 2 (ab56889), Anti-SMCR7 (MID49) (Proteintech, 16413–1-AP).
Novex 4 12 bis tris protein gel
Manufactured by Thermo Fisher Scientific
The Novex™ 4–12% Bis-Tris Protein Gels are pre-cast polyacrylamide gels designed for electrophoretic separation of proteins. They feature a Bis-Tris buffer system and a 4-12% gradient acrylamide concentration for optimal resolution of a wide range of protein sizes.
Automatically generated - may contain errors
Lab products found in correlation
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Total oxphos rodent wb antibody cocktail, by Abcam (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Hrp conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Iblot 7 minute blotting system, by Thermo Fisher Scientific (1 mentions)
Iblot transfer stack, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay kit, by Takara Bio (1 mentions)
4 protocols using novex 4 12 bis tris protein gel
1
Profiling Mitochondrial Dynamics Proteins
2
Protein Extraction and Western Blot Analysis
3
STAT3 Western Blot in Sciatic Nerve
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting of protein extracts from freshly dissected sciatic nerves was conducted with nerve tissue homogenized in 100 µl RIPA buffer (0.5 mM EDTA, 50 mM TrisHCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% Sodium Deoxycholate, 0.1% SDS, pH 8.0) followed by 15 min centrifugation at 6500 rpm, 4 °C. Supernatant was collected and the protein concentration was evaluated by Pierce BCA Assay to prepare the samples with a final concentration of 20 µg/25 µl. Samples and molecular weight marker (Novex Sharp Prestained Protein Standard) were electrophoresed in NuPage Novex 4–12% Bis-Tris Protein Gel and transferred to the nitrocellulose membrane (Novex Gel Transfer Stacks) followed by 1-h blocking using 5% BSA in TBST with 0.1% Tween-20. The primary antibody for STAT3 (1:2000, Cell Signaling) or pSTAT3 (1:2000, Cell Signaling) was diluted in blocking buffer, applied and incubated overnight at 4 °C. After the washing with TBST, membrane was incubated with secondary antibody (Goat anti-rabbit HRP conjugated IgG, 1:5000, Jackson, USA) at room temperature for one hour followed by 2 min incubation ECL Western Blotting Substrate. Imaging of the membrane was performed with the BioRad ChemiDoc Imaging system using the ImageLab software (BioRad) for analysis of the picture. Loading control was obtained by b-actin blotting on stripped membrane.
4
Zn-Induced CHOP and Actin Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Zn-induced expression of CHOP and actin was assessed by Western blotting analysis. GT1-7 cells grown in 6-well culture plates (7.5 × 105 cells per well) were lysed with RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were measured using a Bradford Protein Assay Kit (Takara Bio). Samples were applied to a NuPAGE® Novex 4–12% Bis-Tris Protein Gel and electrophoresed at a constant voltage of 180 V, and then, proteins were transferred to an iBlot™ Transfer Stack (PVDF membranes) using the iBlot® 7-Minute Blotting System (Thermo Fisher Scientific K.K.). Membranes were blocked with 5% nonfat dry milk at room temperature for 1 h and then incubated with rabbit anti-CHOP antibody (1 : 1000 dilution) or mouse anti-actin antibody (1 : 1000 dilution) in 5% BSA, 1x Tris-buffered saline (TBS), and 0.1% Tween-20 overnight. The following day, membranes were incubated with goat anti-rabbit IgG (1 : 2000 dilution) or donkey anti-mouse IgG (1 : 4000 dilution) HRP-conjugated secondary antibodies in 1x TBS containing 0.1% Tween-20 for 1 h, and finally, bands were visualized using SuperSignal™ West Dura Extended Duration Substrate. Band intensities were quantitated using ImageJ software (version 1.39u), and the band intensity of each protein was determined and normalized with respect to actin intensity.
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!