Cells or tissues were lysed in RIPA lysis buffer (P0013B, Beyotime) supplemented with protease inhibitor (4693159001, Roche) and phosphatase inhibitors (4906837001, Roche). Protein concentration was measured using a BCA protein assay kit (23225, Thermo Fisher Scientific). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes using a Trans-Blot Turbo transfer system (Bio-Rad). Membranes were incubated for 1 hour at room temperature in blocking buffer (5% BSA, FA016, GENVIEW), then incubated at 4°C overnight with primary antibodies. Antibodies were incubated with horseradish peroxidase–conjugated secondary antibodies. The primary antibodies are described below. The secondary antibodies were from Promega (W4011, W4021). Signals were detected using ChemiDOC XRS+ and the Image Lab system (Bio-Rad).
Fa016
Manufactured by Genview
Sourced in United Kingdom
The FA016 is a laboratory equipment designed for essential functions required in research and scientific analysis. It features a compact and durable build to support routine operations.
Automatically generated - may contain errors
Lab products found in correlation
Ipvh00010, by Genview (2 mentions)
Pro light chemiluminescence detection kit, by Tiangen Biotech (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Image lab system, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Ab208942, by Abcam (1 mentions)
A0460, by Beyotime (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Protein g agarose beads, by Roche (1 mentions)
5 protocols using fa016
1
Western Blot Protein Analysis
2
Immunohistochemical Analysis of c-Fos in PFC
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Brain sections were incubated with 0.01 M PBS in 0.5% Triton X-100 (Sigma-Aldrich) and 5% bovine serum albumin (FA016, Genview), and then blocked overnight with c-Fos primary antibodies (ab208942, 1:1,000, Abcam, United Kingdom), followed by fluorescent-conjugated secondary antibodies (A0460, 1:500; Beyotime, China) for 2 h. Sections were stained at room temperature with 4,6-diamidino-2-phenylindole dihydrochloride (S2110, Solarbio Life Science & Technology, China). Images of the PFC were acquired with a panoramic scanner and analyzed with ImageJ.
3
Immunofluorescence Staining Protocol
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Cells were fixed with 4% paraformaldehyde after removing the medium and washing three times with PBS. The samples were washed twice with PBS and blocked with blocking buffer (PBS containing 10% normal goat serum (Beyotime, C0265), 1% bovine serum albumin (Genview, FA016) and 0.3% Triton X-100). Antibodies were diluted with blocking buffer and incubated with the samples for 12 hours at 4 °C or 2 hours at RT. Three PBS washes were performed after each time of antibody incubation. Immunofluorescence was detected with a Zeiss LSM800.
4
Protein Separation and Detection via Western Blot
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For western blot assay, proteins were separated on gel where appropriate. And then transferred to PVDF membranes (Millipore, IPVH00010), which were blocked with 5% BSA (Genview, FA016). Next, PVDF membranes were incubated with the indicated antibodies. Lastly, PVDF membranes were incubated with anti-rabbit (Thermo Fisher Scientific, 31460) or anti-mouse (Thermo Fisher Scientific, 31430) secondary antibodies. Western blot results were obtained by digital gel image analysis system (TANON 5500) and Pro-Light chemiluminescence detection kit (TIANGEN, PA112-01).
5
Immunoprecipitation and Western Blot Analysis
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Cells were lysed in NP40 lysis buffer and then cell lysates were centrifuged at 12,000 g, 4 °C for 20 min. 20 µL protein G agarose beads (Roche, 11243233001) were added to the supernatants for preclear. The supernatants were incubated with protein G agarose beads and indicated antibodies at 4 °C for 8 h. Then immunocomplexes were centrifuged at 4 °C for 3 min and NP-40 lysis buffer were used to wash the precipitates three times before subjected to western blot assay.
For western blot assay, proteins were separated on 10% or 12% SDS-PAGE gel where appropriate. Proteins were then transferred to PVDF membranes (Millipore, IPVH00010), which were blocked with 5% BSA (Genview, FA016). Next PVDF membranes were incubated with the indicated antibodies and washed 3 times with TBST (0.05% Tween-20, 150 mM NaCl and 20 mM Tris-HCl). Lastly, PVDF membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (Thermo Fisher Scientific, 31460) or horseradish peroxidase-conjugated anti-mouse (Thermo Fisher Scientific, 31430) secondary antibodies. Western blot results were obtained by digital gel image analysis system (TANON 5500) and Pro-Light chemiluminescence detection kit (TIANGEN, PA112-01).
For western blot assay, proteins were separated on 10% or 12% SDS-PAGE gel where appropriate. Proteins were then transferred to PVDF membranes (Millipore, IPVH00010), which were blocked with 5% BSA (Genview, FA016). Next PVDF membranes were incubated with the indicated antibodies and washed 3 times with TBST (0.05% Tween-20, 150 mM NaCl and 20 mM Tris-HCl). Lastly, PVDF membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (Thermo Fisher Scientific, 31460) or horseradish peroxidase-conjugated anti-mouse (Thermo Fisher Scientific, 31430) secondary antibodies. Western blot results were obtained by digital gel image analysis system (TANON 5500) and Pro-Light chemiluminescence detection kit (TIANGEN, PA112-01).
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