Protein lysates (10 μg) were incubated with nonreducing 2× Novex tris-glycine SDS sample buffer (Thermo Fisher Scientific) for 30 min at RT and then separated in Novex 10% gelatin protein gels (Thermo Fisher Scientific) in 1× Novex tris-glycine SDS running buffer (Thermo Fisher Scientific). After electrophoresis, the gels were washed twice for 15 min at RT with 1× Novex renaturing buffer (Thermo Fisher Scientific) and then washed for 30 min at RT with 1× Novex developing buffer (Thermo Fisher Scientific). After the gels were incubated with fresh developing buffer for 2 days at 37°C, the gels were stained with Coomassie blue for 1 hour at RT. The gels were destained in 30% methanol until the bands appeared. Quantification of digested bands from scanned gels was performed using Fiji (50 (link)). Original scans of gels can be found in data file S1.
Novex renaturing buffer
Manufactured by Thermo Fisher Scientific
Novex renaturing buffer is a reagent used in protein refolding and renaturation processes. It helps facilitate the proper folding of denatured or misfolded proteins to restore their native structural and functional characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Novex tris glycine sds running buffer, by Thermo Fisher Scientific (1 mentions)
Novex 10 zymogram plus gelatin protein gel, by Thermo Fisher Scientific (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Developing buffer, by Thermo Fisher Scientific (1 mentions)
Mmp 9, by R&D Systems (1 mentions)
2 protocols using novex renaturing buffer
1
Protein Gelatin Zymography Assay
2
Visualizing and Quantifying MMP Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Zymography is a conventional method for detecting MMP activity. To confirm MMP inhibition in gelatin zymography we used purified human MMP2 or MMP9 enzyme. After running the zymogram with human MMP2, the gel lanes were cut and incubated with engineered TIMP3 variants during development in a separate container overnight at 37°C before staining. For tissues, cardiac LV tissues were homogenized in RIPA Buffer Solution (Teknova, Hollister, CA) supplemented with PMSF. Tissue lysate (50 μg) or recombinant human MMP2, or MMP9 (10 ng, R&D Systems, Minneapolis, MN) was loaded per lane onto Novex 10% Zymogram Plus (Gelatin) Protein Gel (Thermo Fisher Scientific, Waltham, MA). After electrophoresis, gels were incubated with Novex Renaturing buffer (Thermo Fisher Scientific). Gels were then incubated overnight in Developing buffer (Thermo Fisher Scientific) with or without TIMP3 molecules. The following day, gels were stained with SimplyBlue Safestain (Thermo Fisher Scientific). MMP activity was quantified using ImageJ software.
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!