RNA immunoprecipitation (RIP) assay was performed to further confirm the direct association between lncRNA MIAT and miR-613. Magna RIP™ RNA Immunoprecipitation kit (EMD Millipore) was used by following the manufacturer's protocols. Briefly, the LSCC cells were transfected with the miR-613 mimics or miR-NC (50 nM) for 48 h before being lysed using the RIP lysis buffer (EMD Millipore). A total of 50 µl magnetic beads were resuspended in 100 µl RIP washing buffer and swirled. After the supernatant had been discarded, magnetic beads were resuspended with 100 µl RIP washing buffer, and 5 µg antibodies were added. The magnetic bead-antibody complex was washed and resuspended in 900 µl RIP washing buffer before incubation with cell lysates. The cell lysates were then incubated with the RIP buffer containing magnetic bead-antibody complex at 4˚C overnight. Finally, co-precipitated magnetic bead-protein complex and input were separately detached with proteinase K to extract RNA for subsequent RT-qPCR detection of MIAT. The antibodies used for RIP were rabbit anti-human Ago2 (1:500; Abcam; cat. no. ab186733) and rabbit anti-human IgG (1:500; Abcam; cat. no. ab109489).
Rabbit anti human igg
Manufactured by Abcam
Rabbit anti-human IgG is a secondary antibody that recognizes and binds to human immunoglobulin G (IgG) molecules. It is produced by immunizing rabbits with human IgG and can be used in various immunoassays and detection techniques.
Automatically generated - may contain errors
2 protocols using rabbit anti human igg
1
Validating lncRNA MIAT-miR-613 Interaction
2
Infliximab Binding to Recombinant TNF-α
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Binding of IFX to recombinant (r)TNF‐α was studied using standard denaturing Western blot techniques. Briefly, 2‐mercaptoethanol and heat‐treated rTNF‐α was loaded onto a preprepared iBlot sodium dodecyl sulphate (SDS) denaturing gel (Novex; Life Technologies, Paisley, UK) in denaturing SDS running buffer (NuPAGE Tris‐Acetate SDS; Invitrogen Life Technologies). After transferring to a nitrocellulose membrane, the gel was blocked (5% fish gelatin for 1 h, shaking), washed [1% phosphate‐buffered saline (PBS)‐Tween20 ×4] and primary antibodies added [infliximab, 2 μg/ml; polyclonal rabbit anti‐mouse TNF‐α (Abcam ab9739; Abcam, Cambridge, UK), 10 μg/ml]. Membranes were incubated for 16 h at 4°C, washed and secondary antibodies [rabbit anti‐human IgG, 1 μg/ml (Abcam); goat anti‐rabbit, 2 μg/ml (Abcam)] were added (1 h with shaking). Membranes were then washed and treated with ECL (1 min; Promega, Southampton, UK) before imaging using Bio‐Rad Chemidoc (Bio‐rad, Hertfordshire, UK). Data were processed using Image Lab Software (Bio‐rad).
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