Cell extracts were lysed in 10x RIPA solution (Merck Millipore) and or mouse striatal punches were homogenized in a urea/DTT solution, both containing a cocktail of protease inhibitors (Roche pharmaceuticals), followed ultrasound sonication. Protein concentration levels were determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific) for cell lysates and using NZYBradford reagent (Nzytech) for mouse samples. Sixty to eighty protein µg were resolved in SDS-polyacrylamide gels (7.5% and 12%). Followed by protein transfer to PVDF membrane (Merck Millipore) and antibody probing using the following antibodies: mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences); mouse anti-IC2 (polyQ chain) (1:1000, ref. MAB1574, Merck Millipore); rabbit anti-LC3B (1:1000, ref. NB100-2220, Novus); rabbit anti-SQSTM1 (1:1000, ref. 5114S, Cell Signaling); mouse anti-β-actin (1:5000, ref. A5316, Sigma Aldrich); rabbit anti-DARP-32 (1:1000, ref. AB10518, Merck Millipore). Optical densiometric analysis was carried out using Image J software (USA).
Nzybradford reagent
Manufactured by NZYTech
The NZYBradford reagent is a colorimetric assay used to determine the concentration of total protein in a sample. It is a simple, rapid, and sensitive method for protein quantification.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (1 mentions)
Rabbit anti sqstm1, by Novus Biologicals (1 mentions)
Rabbit anti lc3b, by Merck Group (1 mentions)
Mouse anti β actin, by Cell Signaling Technology (1 mentions)
Mouse anti ataxin 2, by BD (1 mentions)
2 protocols using nzybradford reagent
1
Analysis of Ataxin-2 and Autophagy Markers
2
Immunoblotting Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were either lysed in 1x RIPA solution (Merck Millipore), when using cell extracts or homogenized in a urea/DTT solution, when using mouse striatal punches, both containing a cocktail of protease inhibitors (Roche). Following, samples were further submitted to 5 cycles of ultrasound sonication of 30 sec ON, 30 sec OFF, 5 cycles (Bioruptor Pico). Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scienti c) for cell lysates and the NZYBradford reagent (Nzytech) for mouse samples. Equal amounts of protein extracts were resolved in SDS-polyacrylamide gels (7.5% and 12%), followed by protein transfer to PVDF membranes (Merck Millipore), membrane blocking in 0.1% (v/v) Tween-20/TBS containing 3% of BSA or 5% of milk, and antibody probing overnight at 4 ºC for primary and 2h at room temperature for secondary. The following antibodies were used: mouse anti-
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!