The APEX2 reaction was performed as described above and lysed in RIPA. Aliquots were taken for input, post-streptavidin flow-through, and StrePD. Proteins were separated on an SDS-PAGE gel and transferred to nitrocellulose for Western blotting with the indicated reagents/antibodies. Detection reagents used were streptavidin-HRP (1:1,000; GE Healthcare; RPN1231V), rabbit anti-lamin-B1 (1:10,000; Abcam; ab16048), mouse anti-lamin-A/C (1:5,000; Active Motif; 39287), rabbit anti-emerin (1:5,000; Santa Cruz; sc-15378), mouse anti-SC-35 (1:2,500, Sigma-Aldrich; S4045), rabbit anti-HNRNPA1 (1:2,500; ProteinTech; 11176-1-AP), and rabbit anti-SRSF1 (1:2,500; ProteinTech; 12929-2-AP). Imaging for streptavidin-HRP was done on a Licor Odyssey Fc machine. All other Western blots were imaged with a Licor Odyssey CLx machine.
Streptavidin hrp
Manufactured by LI COR
Streptavidin-HRP is a conjugate of the protein streptavidin and the enzyme horseradish peroxidase (HRP). It is used as a detection reagent in various bioanalytical techniques, such as Western blotting, ELISA, and immunohistochemistry, to amplify and visualize the signal of biotinylated targets.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti lamin a c, by Active Motif (1 mentions)
Odyssey clx machine, by LI COR (1 mentions)
Rabbit anti lamin b1, by Abcam (1 mentions)
Odyssey fc machine, by LI COR (1 mentions)
Streptavidin hrp, by GE Healthcare (1 mentions)
Rabbit anti emerin, by Santa Cruz Biotechnology (1 mentions)
Mouse anti sc35, by Merck Group (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
2 protocols using streptavidin hrp
1
Biotinylated Protein Identification via Immunoblotting
2
Cytokine Profiling in Aging Mice
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Cytokine levels in supernatants from PM of adult and old mice were measured using either an enzyme‐linked immunosorbent assay (ELISA) or by cytokine arrays. All materials were purchased from R&D systems (IL‐1beta/IL‐1F2: DY401‐05; IL‐1ra/IL‐1F3: DY480; IL‐6: DY406; IL‐10: DY417 05; IL‐12 p70: DY419 05; TGF‐beta‐1: DY1679‐05; TNF‐alpha: DY410‐05; Proteome Mouse XL Cytokine Array Kit: ARY028). Assays were performed according to the manufacturer instructions for the ELISAs. For cytokine arrays, samples from all biological replicates of an experimental group were pooled and used for the assay. Detection of the array spots was done with fluorescent‐labelled Streptavidin‐HRP (LI‐COR Biosciences, 926‐32230), and arrays were analyzed using an Odyssey infrared imager (LI‐COR Biosciences). Densitometric analysis of array spots and background subtraction was performed using manufacturers software (Table S4 ).
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