Purified recombinant proteins were separated by SDS-PAGE (10%) using the Mini-Protean system III (Bio-Rad, Hercules, CA, USA) and stained with Coomassie Brilliant Blue. For Western blot analysis, recombinant proteins were transferred onto PVDF membranes (GE Healthcare, Chicago, IL, USA) using an Electro Transfer Unit (Bio-Rad). The membranes were incubated with either mouse anti-6xHIS monoclonal antibody (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA), mouse polyclonal antibodies against rSAG1m (1:1000) or sera from NbHsp90.3-SAG1HC immunized mice (1:100) as primary antibodies. Then, membranes were incubated with alkaline phosphatase conjugated goat anti-mouse IgG (1:5000, Sigma, St. Louis, MO, USA). The reaction was developed by the addition of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Promega) substrate. PageRuler™ Prestained Protein Ladder (Fermentas, Waltham, MA, USA) was used as molecular marker.
Mouse anti 6xhis monoclonal antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Mouse anti-6xHIS monoclonal antibody is a laboratory reagent used for the detection and purification of proteins tagged with a 6xHIS (Hexahistidine) sequence. This antibody specifically binds to the 6xHIS tag, allowing for the identification and isolation of the target protein in various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by GE Healthcare (2 mentions)
Electro transfer unit, by Bio-Rad (2 mentions)
Nbt bcip, by Promega (2 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (2 mentions)
Mini protean 3 system, by Bio-Rad (1 mentions)
Alkaline phosphatase conjugated goat anti mouse igg, by Merck Group (1 mentions)
2 protocols using mouse anti 6xhis monoclonal antibody
1
Recombinant Protein Characterization by SDS-PAGE and Western Blot
2
Western Blotting of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as previously described (Corigliano et al., 2013) .
Briefly, recombinant proteins previously separated by SDS-PAGE 15%, were transferred onto PVDF membranes (GE Healthcare, Buckinghamshire, UK) using an Electro transfer Unit (Bio-Rad). Firstly, the membranes were incubated with mouse anti-6XHIS monoclonal antibody (1:1,000, Cell Signaling Technology Inc., MA, USA), sera from mice experimentally infected with N. caninum 2 x 10 6 tachyzoites from Nc-1 strain (dilution, 1:500), mouse anti-rNcSAG1 polyclonal antibody (1:500) or mouse anti-rAtHsp81.2 polyclonal antibody (1:500) (Corigliano et al., 2011) , as primary antibodies. Later, the membranes were incubated with alkaline phosphatase conjugatedgoat polyclonal anti-mouse IgG (complete molecule) secondary antibody (1:5,000, SIGMA, MO, USA). After washing, the reaction was developed by the addition of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Promega, WI, USA) substrate. PageRuler TM Prestained Protein Ladder (Fermentas, MA, USA) was used as molecular marker.
Briefly, recombinant proteins previously separated by SDS-PAGE 15%, were transferred onto PVDF membranes (GE Healthcare, Buckinghamshire, UK) using an Electro transfer Unit (Bio-Rad). Firstly, the membranes were incubated with mouse anti-6XHIS monoclonal antibody (1:1,000, Cell Signaling Technology Inc., MA, USA), sera from mice experimentally infected with N. caninum 2 x 10 6 tachyzoites from Nc-1 strain (dilution, 1:500), mouse anti-rNcSAG1 polyclonal antibody (1:500) or mouse anti-rAtHsp81.2 polyclonal antibody (1:500) (Corigliano et al., 2011) , as primary antibodies. Later, the membranes were incubated with alkaline phosphatase conjugatedgoat polyclonal anti-mouse IgG (complete molecule) secondary antibody (1:5,000, SIGMA, MO, USA). After washing, the reaction was developed by the addition of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Promega, WI, USA) substrate. PageRuler TM Prestained Protein Ladder (Fermentas, MA, USA) was used as molecular marker.
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!