ALKBH5 RIP was carried out using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Sigma, 17–700) according to the manufacturer’s protocol. 3.5 μg of anti-ALKBH5 antibody (Sigma, HPA007196) was used for each reaction, with the same amount of IgG used as a control.
Protocol detail
ALKBH5 RNA Immunoprecipitation
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Corresponding Organization : The University of Texas Health Science Center at San Antonio
Other organizations : Houston Methodist, The University of Texas at San Antonio, National Cancer Institute, Center for Cancer Research
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Variable analysis
independent variables
- Use of anti-ALKBH5 antibody (Sigma, HPA007196) for immunoprecipitation
- RNA-binding proteins that interact with ALKBH5
- Use of the same amount of IgG as a control for immunoprecipitation
- Positive control: Immunoprecipitation with anti-ALKBH5 antibody
- Negative control: Immunoprecipitation with IgG
Annotations
Based on most similar protocols
According to Protocol 1, the RIP assay used 2 μg of ALKBH5, FTO, or Rabbit IgG control antibodies. This suggests that the optimal antibody amount for the RIP assay may be around 2 μg per reaction.
The Input protocol and Protocol 1 both used a Magna RIP RNA-Binding Protein Immunoprecipitation Kit, indicating that this kit is a suitable choice for the RIP assay.
Protocol 1 mentions that the coprecipitated RNAs were detected by qRT-PCR, which could be a useful validation step to confirm the method's accuracy and specificity.
The Input protocol used 3.5 μg of anti-ALKBH5 antibody, which is slightly higher than the 2 μg used in Protocol 1. This suggests that the optimal antibody amount may need to be empirically determined for each specific experiment.
According to Protocol 1, the sensitivity and specificity of the RIP assay can be assessed by using appropriate positive and negative controls, such as the Rabbit IgG control antibody.
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