To isolate the soluble cytoplasm and insoluble membrane, the 35S:MfSTMIR‐FLAG fusion protein was transiently expressed in N. benthamiana and extracted as described previously (Lei et al., 2015 ). The protein concentrations from the soluble or insoluble fractions were measured using the Coomassie (Bradford) protein assay kit and were adjusted to equal concentrations. The fractions (1 mg) were analyzed by 10% SDS‐PAGE and then immunoblotted using Flag monoclonal antibody (F3165 Sigma). Next, a 1:5000 dilution of H+‐ATPase (Agrisera) and a 1:5000 dilution of cFBPase (Agrisera) were used as plasma membrane and cytosolic markers, respectively.
Flag monoclonal antibody
Manufactured by Merck Group
The Flag monoclonal antibody is a laboratory tool used to detect and purify recombinant proteins. It recognizes a specific amino acid sequence, allowing for the identification and isolation of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using flag monoclonal antibody
1
Subcellular Fractionation and Protein Detection
2
Western Blot Antibody Validation
3
Immunoprecipitation and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in NP-40 lysis buffer (20mM Tris-HCl pH8.0, 150mM NaCl, 1% Nonident P-40, 1mM PMSF) for 30min on ice. Extracts were centrifuged at 13,000rpm for 10min at 4°C, and the protein concentration was measured using the Bradford assay. Each cell lysate (1.5mg) was incubated with CLU polyclonal antibody (Santa cruz) or flag monoclonal antibody (Sigma) for overnight at 4°C. Following incubation, protein was immunoprecipitated using protein A/G agarose beads (Santa cruz) for 3hr at 4°C with gently rotation. The immunoprecipitates was washed three times with lysis buffer and boiled in 40°C of 1X SDS sample buffer for 5min at 95°C. After centrifugation, the supernatant was analyzed by Western blot.
4
Quantum Dot Antibody Complex Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following titration experiment results, all experiments in slices were performed at a dilution of approximately 1 QD to 10 anti-GFP. QD-antibody complexes were prepared by incubating 3 μl of QD655 Goat F(ab')2 anti-Rabbit IgG (Molecular Probes, #Q-11421MP) with either 3 μl of GFP Rabbit Serum Polyclonal Antibody (Molecular Probes, #A6455) or 3 μl of FLAG Monoclonal Antibody (Sigma-Aldrich, #2555). The cocktail was then diluted in 54 μl of PBS (final QD concentration of 50 nM), vortexed, briefly spun down and incubated for 30 min at room temperature.
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!