Purity and molecular mass of the recombinant proteins were checked by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method using a discontinuous buffer system as described before [59 (link)]. About 10 μg of the purified protein was loaded onto the SDS gel (12% acrylamide) and electrophoresis was performed at 120 V for about 60 min. The molecular weight (MW) of proteins was estimated by comparison with the standard protein ladder (MW protein marker range 10–180 kDa, Page Ruler, ThermoFisher). To estimate the size of the enzyme/protein complex, native PAGE was performed using Mini-Protean TGX Precast Gels (Bio-Rad) with a polyacrylamide gradient of 4–15%; Amersham High Molecular Weight Calibration Kit (GE Healthcare) was used as a reference, and gels were run with native-gel running buffer (192 mM Glycine, 25 mM Tris/HCl pH 8.8; without SDS) under constant current of 8 mA per gel for 3 h [63, 64]. Protein bands were visualised by staining the gel with InstantBlue (Ultrafast Protein Stain, Sigma Aldrich). For protein identity, bands were excised from the stained gels and processed for mass spectrometry analysis as described before [24 , 26 (link)].
Instantblue ultrafast protein stain
Manufactured by Merck Group
Sourced in United States
InstantBlue Ultrafast Protein Stain is a ready-to-use, one-step protein stain solution designed for rapid and sensitive detection of proteins in polyacrylamide gels. It provides fast, high-quality staining with a simple workflow.
Automatically generated - may contain errors
Lab products found in correlation
Pageruler, by Thermo Fisher Scientific (1 mentions)
Amersham high molecular weight calibration kit, by GE Healthcare (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Pierce f ab 2 preparation kit, by Thermo Fisher Scientific (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Multiscreen htsip filter plates, by Merck Group (1 mentions)
Np osu, by Biosearch Technologies (1 mentions)
5 protocols using instantblue ultrafast protein stain
1
Protein Purity and Molecular Mass Analysis
2
SDS-PAGE Analysis of A3V Vectors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The purity of the A3V vectors was assessed via SDS-PAGE analysis. A total of 10 μL of purified vector was denatured in reducing sample buffer by incubation at 95°C for 5 minutes, followed by separation on a 9% SDS-PAGE polyacrylamide gel for 80 minutes at 140 V. Gels were stained with InstantBlue Ultrafast Protein Stain (catalog no. ISB1L-1L; Sigma-Aldrich) overnight to visualize capsid proteins.
3
Pepsin Digestion of Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One milligram of antibody (in-house-produced FcγRI hybridoma clones 1E2 and 1E3 and antibodies 197 and 10.1) was digested with pepsin using the Pierce F(ab’)2 preparation kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol. To determine purity, F(ab’)2 preparations were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (reducing condition, 4–20% gradient gel; Bio-Rad, Hercules, CA, USA) and stained using InstantBlue Ultrafast Protein Stain (Sigma-Aldrich, Burlington, MA, USA).
4
SDS-PAGE Analysis of BVZ Stability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were diluted with a 2× sample buffer (0.125 M Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue, and 5% beta-mercaptoethanol) and were separated by SDS-PAGE 10% polyacrylamide gel. PageRuler Prestained Protein Ladder (Thermofisher,
To determine the BVZ stability over time, the SDS-PAGE of the samples was repeated for up to 90 days.
To determine the BVZ stability over time, the SDS-PAGE of the samples was repeated for up to 90 days.
5
ELISA and ELISPOT Protocols for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Enzyme-linked immunosorbent assays (ELISAs) were done as described (48 (link)) with addition of 0.05% Tween 20 in block and wash buffers. 4-Hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to BSA (NP-BSA, Ratio 28) was generated in-house with BSA fraction V (Roth Cat# 8076.3) and NP-OSu (Biosearch Technologies Cat# N1010-100). Plates were coated with 2 µg/ml NP-BSA or 1 µg/ml anti-light chain antibodies and developed with 1 µg/ml anti-isotype antibodies and the standards listed in Supplementary Table 1
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!