The VAPA knockdown by the Trim-Away method was performed as previously described with some modifications46 (link),47 (link). In brief, anti-VAPA antibody was concentrated using Amicon Ultra-0.5 100 KDa centrifugal filter devices (Millipore) and replaced the buffer with PBS. Mouse GV oocytes were injected with anti-VAPA antibody (final concentration of 1 μg/μl) and anti-mouse antibody (final concentration of 5 μg/μl) and kept 1 h in IBMX-containing M16 medium. Then GV oocytes were injected with Trim21 mRNA (1000 ng/μl) for 3–5 hours to allow protein expression. For the control experiment, normal mouse immunoglobulin G (IgG) antibody was used instead of anti-VAPA antibody.
Amicon ultra 0.5 100 kda centrifugal filter devices
Manufactured by Merck Group
The Amicon Ultra-0.5 100 KDa centrifugal filter devices are a type of lab equipment used for the separation and concentration of macromolecules, such as proteins, from solutions. The devices utilize a centrifugal force to pass the sample through a semi-permeable membrane, which retains molecules larger than the specified molecular weight cut-off (100 kDa in this case) while allowing smaller molecules to pass through.
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Lab products found in correlation
2 protocols using amicon ultra 0.5 100 kda centrifugal filter devices
1
Trim-Away VAPA Knockdown in Mouse Oocytes
2
Anti-AGO2 Antibody Microinjection
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The anti-AGO2 antibody used was rabbit anti-AGO2 (Abcam, ab32381), and purchased in azide-free format and concentrated using Amicon Ultra-0.5 100 kDa centrifugal filter devices (Millipore) to remove traces of azide and replace the buffer with PBS. Prior to microinjection, antibodies were diluted in 1× PBS containing Ago2-siRNA (100 µM) to the following concentrations: anti-AGO2 (0.5 mg/ml) and Ago2-siRNA (10 µM). Prior to microinjection, antibodies-siRNA mixture was incubated on ice for 30 min. And, microinjection was performed using a micromanipulator and Eppendorf Femtojet system mounted on the OLYPUS microscope. Once the glass needle was transiently inserted into the cytoplasm, the needle was quickly withdrawn when slight swelling in cytoplasm appears, indicating the successful injection.
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