Tissue samples were homogenized in 1% SDS processing buffer and protein was quantified using the Bio-Rad protein DC assay (Bio-Rad Laboratories, Berkeley, CA, USA). Forty μg of total protein was loaded onto a 12–15% polyacylamide gradient gel (Mini-PROTEAN TGX Precast Mini Gel, Bio-Rad Laboratories) and transferred to a nitrocellulose membrane. Membranes were blocked in 5% non-fat dried milk or BSA (phospho-FMRP) in TBS, incubated overnight in primary antibodies against PSD-95 (1:1,000; Cell Signaling, Beverly, MA, USA), SAPAP-3 (1:500, ProteinTech, Chicago, IL, USA), FMRP (1:500, Millipore, Billerica, MA, USA), phospho-FMRP (1:1,000; Abcam, Cambridge, MA, USA), and GAPDH (1:30,000; Millipore) diluted in TBS + 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA). The following day, membranes were incubated for 1 hr in the appropriate secondary antibodies (1:20,000; Li-Cor Biosciences, Lincoln, NE, USA) and then imaged using the Odyssey imaging system (LiCor Biosciences).
Sapap 3
Manufactured by Proteintech
Sourced in United States
SAPAP-3 is a protein that is a member of the SAPAP family. It is a critical component of the postsynaptic density in neurons and plays a role in the organization and function of the postsynaptic scaffold.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (1 mentions)
Gapdh, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
Glun2a, by Merck Group (1 mentions)
Glun2b, by Merck Group (1 mentions)
Glun1, by Abcam (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
Amersham ecl, by Cytiva (1 mentions)
2 protocols using sapap 3
1
Western Blot Analysis of Synaptic Proteins
2
Western Blot Analysis of Subcellular Fractions
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Subcellular fractions were obtained as previously reported [26 (link), 27 (link)]. Equivalent amounts (total proteins, 50 μg; subcellular fractions, 10 μg) of the samples were separated on 10–12% SDS-PAGE gels and then transferred onto PVDF membranes. The membranes were incubated with 5% nonfat milk for 1 h at room temperature. The target proteins were blotted with primary antibodies against SAPAP3 (1:200, Proteintech), GluN1 (1:1000, Abcam), GluN2A (1:1000, Millipore), GluN2B (1:1000, Millipore), and GAPDH (1:5000, Abcam) overnight at 4 °C. On the following day, the blots were incubated with HRP-conjugated secondary antibodies (1:3000, room temperature for 1 h). An enhanced chemiluminescence detection system (Amersham ECL) and the Bio-Rad ChemiDoc XRS + system were used to detect and quantify the bands. Densitometry was performed using Quantity One Software (Bio-Rad Laboratories, Hercules, USA). The total protein samples were normalized to GAPDH expression, and the proteins in the subcellular fractions were normalized to the total protein samples.
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