Co-IP was performed as previously described [23 (link)] using antibodies for tau (Tau5 EMD Millipore), actin (Sigma-Aldrich), RPL28 (GeneTex), or RPP0 (Gene-Tex). Western blots were performed as previously described [24 (link)]. Briefly, 20 μg of protein were denatured by mixing with a sample buffer (consisting of 2x Laemmli buffer with 5% β-mercaptoethanol) and subjecting to heat at 100°C for 10 min. Samples were loaded in 10% tris-glycine gels (Life Technologies). Gels were subjected to 100 mV until the dye front reached the bottom of the gel. We then performed wet transfers as indicated previously [25 (link)]. Samples were blocked in 7% non-fat dry milk (LabScientific) with 0.02% sodium azide. This milk was used to mix antibodies for incubation with the membranes. Antibodies used were anti total tau h-150 (1:1000) from Santa Cruz Biotechnology, actin (1:1000) from Sigma, RPL28 from GeneTex, RPP0 from GeneTex, and calnexin C-20 from Santa Cruz Biotechnology.
Rpl28
Manufactured by GeneTex
RPL28 is a protein that is a component of the 60S ribosomal subunit. It is involved in the translation process, which is the synthesis of proteins from messenger RNA (mRNA). RPL28 plays a crucial role in the assembly and function of the ribosome.
Automatically generated - may contain errors
Lab products found in correlation
Actin, by Cell Signaling Technology (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Tris glycine gel, by Bio-Rad (1 mentions)
Phospho rps6ser240 244, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Actin, by Merck Group (1 mentions)
2 protocols using rpl28
1
Co-Immunoprecipitation and Western Blot Analysis
2
Western Blot Analysis of Tau Proteins
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Western blot experiments were performed as described previously [26 (link)]. Sample lysate protein concentrations were normalized with lysis buffer and denatured with 4 × Laemmli buffer with 10% β-mercaptoethanol. Proteins were resolved in 10% Tris–Glycine gels (BioRad) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH00010). Membranes were blocked in 1X PBS with 0.1% Tween-20 (PBS-T). All antibodies were diluted in 5% milk or 5% BSA in PBS-T. Primary antibodies were used as follows: PHF1 (1:2000, generously provided by Dr. Peter Davies), H150 total tau (1:2000, SantaCruz), Tau 5 total tau (1:2000, Millipore), actin (1:5000, Cell Signaling Technology), GAPDH (1:5000, Cell Signaling Technology), RPL28 (1:1000, GeneTex), EIF3E (1:1000, Sigma-Aldrich), Phospho-RPS6 Ser240/244 (1:1000, Cell Signaling Technology), total RPS6 (1:1000, SantaCruz). Bands were detected using ECL (GE Amersham Imager 600) using SuperSignal West Pico (Thermo Fisher, 1863096). Blot images were quantified using ImageJ (1.52b) and normalized to either GAPDH or β-actin.
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