Briefly, 350 μL of an overnight culture of STM sopB::FLAG, STM invF::Tn5 sopB::FLAG and STM sopB::FLAG transformed with pINIIIA1, pLAX185, pLAD235 and pLAD256 were inoculated in flasks with 10 mL of LB-Miller complemented with IPTG 1 mM, the cultures were incubated for 3.5 h at 37 °C in agitation (225 rpm) and the optical density was measured at 600 nm. The cultures were centrifugated for 20 min at 11,000 × g at 4 °C, the supernatants were discarded, then 100 μL of Laemmli buffer were added to the pellets and were heated at 95 °C for 10 min. Samples were loaded onto a 12% SDS-PAGE and stained with Coomassie stain. Western blot was performed by transferring the proteins to a PVDF membrane (Merck) and using anti-FLAG-HRP (1:5,000), anti-GroEL(1:10,000) antibodies (Abcam) and recombinant protein G-HRP. Membrane development was done as described above.
Anti flag hrp
Manufactured by Abcam
Anti-FLAG-HRP is a horseradish peroxidase (HRP) conjugated antibody that specifically recognizes the FLAG epitope tag. It can be used for the detection of proteins fused with the FLAG tag in various applications such as Western blotting, immunoprecipitation, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti foxo3, by Cell Signaling Technology (1 mentions)
Anti survivin, by R&D Systems (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit and goat anti mouse secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
Anti dna pkcs, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Abcam (1 mentions)
2 protocols using anti flag hrp
1
SopB Protein Expression Analysis
2
Ionizing Radiation Induced DNA-PKcs Signaling
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SW480 cells were transfected with survivin/PI3K expression vectors for 48 hours and cell lysates were prepared at 1 hour after a 4 Gy irradiation. Equal amounts of lysates were added to coupled Dynabeads Protein G (Thermo Fisher Scientific) and antibody complexes: Anti-GFP (#ab290; Abcam, RRID:AB_303395), anti-Flag (#2368S; Cell Signaling Technology), anti-DNA-PKcs (#MS-369-P1; Thermo Fisher Scientific) or IgG (Mouse #SC-2025, Rabbit #SC-2027; Santa Cruz Biotechnology) and incubated in a rotating mixer at 5 rpm overnight at 4 C. Next, beads were washed with PBS and proteins were eluted by boiling, subjected to Western immunoblotting and detected with primary antibodies: anti-survivin (AF886; R&D Systems), Anti-GFP (ab290; Abcam), anti-DNA-PKcs (#MS-369-P1; Thermo Fisher Scientific), anti-Flag-HRP (#ab49763; Abcam), anti-b-actin (A5441-.2ML; Sigma-Aldrich), anti-Foxo3 (#2497S), or anti-Foxo3 pS253 (#9466S; Cell Signaling Technology). For detection, horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Santa Cruz Biotechnology) and LI-COR WesternSure Premium Chemiluminescent Substrate (LI-COR) were used.
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