The CDS region of MoCBP7 was fused into the prokaryotic expression vector pET21a with a 3×FLAG tag, and the CDS region of MoCNB1 was fused into the prokaryotic expression vector pGEX4T with a GST tag. The primers can be obtained in Table S2 , and the fusion proteins were expressed in the Escherichia coli strain BL21 (DE3) using IPTG (isopropyl β-D-1-thiogalactopyranoside) as an inducer. After 16 h of induction, the cells were enriched and resuspended with binding buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 10 mM Imidazole), followed by sonication to break up the cells. Protein supernatants containing GST–MoCnb1 and GST proteins were co-incubated with GST beads (BBI, Shanghai, China) for 2 h at 4 °C. After five washes with the wash buffer (BBI, Shanghai, China), protein supernatants containing 3 × FLAG–MoCbp7 protein were co-incubated with beads again for 2 h at 4 °C. The proteins were eluted using elution buffer (BBI, Shanghai, China) and detected by immunoblotting with an anti-GST antibody and an anti-FLAG antibody (HUABIO, Hangzhou, China).
Anti flag antibody
Manufactured by Huabio
Sourced in China
The Anti-FLAG antibody is a laboratory reagent used for the detection and purification of proteins that have been tagged with the FLAG peptide sequence. The antibody specifically binds to the FLAG tag, allowing for the identification and isolation of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using anti flag antibody
1
Recombinant Protein Expression and Purification
2
Phos-tag Assay for Phosphorylated Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Phos‐tag assays, the transformants expressing CreA‐3×FLAG or Crf1‐3×FLAG were cultured in CM for 2 days and then transferred to MM or MM−C supplemented with 1% l ‐arabinose for 6 h. The proteins were extracted with a lysis buffer containing 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1% Triton X‐100, and 1% protease inhibitor cocktail (Bio Basic I). When necessary, 1% phosphatase inhibitor cocktail 1 and 2 (ApexBio) was added into samples during protein extraction. Samples were resolved in 8% SDS‐polyacrylamide gels containing 100 μM acrylamide‐pendant Phos‐tag ligand (Wako) and 200 μM MnCl2 or ZnCl2. Gels was run at 25 mA for 2–3 h. Gels were then equilibrated in the transfer buffer containing 5 mM EDTA for 30 min and in the transfer buffer without EDTA for 10 min. Proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane at 300 mA for 2 h at 4°C. The proteins were detected using an anti‐FLAG antibody (Huabio). The proteins were also detected by normal western blot.
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!