Adrenal glands and brain extracts were collected from P0-1 Syt-7 WT and KO mice and lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Invitrogen, 89900). The supernatants were collected and protein concentrations were estimated by using the BCA Protein Assay Kit (Pierce 23227) and plotting the resulting BSA curve. 25 μg and 15 μg of protein, from adrenal glands and brain extracts, respectively, were resolved by 4–12% SDS-PAGE (Invitrogen, Thermo Fisher Scientific) and wet-transferred onto an Amersham Hybond LFP PVDF membrane (GE Healthcare). The membrane was blotted with rabbit polyclonal α-Syt7 (1:500; Synaptic Systems SY105173) and mouse monoclonal α-VCP (1:10000; Abcam ab11433), as a loading control, followed by HRP-conjugated α-rabbit (1:2000; Agilent Dako-P0448) and HRP-conjugated α-mouse (1:10000; Agilent Dako-P0447) secondary antibodies. The blot was developed by chemiluminescence SuperSignal West Femto and Pierce ECL Plus Western blotting substrate systems (Thermofisher Scientific) and immunoreactive bands were detected using the FluorChemE image acquisition system (ProteinSimple) equipped with a cooled CCD camera.
Amersham hybond lfp pvdf membrane
Manufactured by GE Healthcare
Amersham Hybond LFP PVDF membrane is a polyvinylidene fluoride (PVDF) membrane used in various laboratory applications. It is designed for effective protein transfer and immobilization in Western blotting and other protein analysis techniques.
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4 protocols using amersham hybond lfp pvdf membrane
1
Syt7 Expression in Adrenal Glands and Brain
2
Quantifying Ubiquitous Munc13 Isoforms
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Extracts from HEK293FT cells expressing ubMunc13-2-EGFP or Munc13-1-EGFP, as well as non-expressing cells, were collected and lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Invitrogen, 89900). The supernatants were collected and protein concentrations were estimated using the BCA Protein Assay Kit (Pierce 23227) after plotting the resulting BSA curve. 25 mg of protein was resolved by 4–12% SDS-PAGE (Invitrogen, Thermo Fisher Scientific) and wet-transferred onto an Amersham Hybond LFP PVDF membrane (GE Healthcare). The membrane was blotted with rabbit polyclonal α-GFP (1:300; Synaptic Systems SY132003) and HRP-conjugated mouse monoclonal α-β-Actin-Peroxidase (1:10000; Sigma-Aldrich A3854), as a loading control, followed by HRP-conjugated α-rabbit (1:2000; Agilent Dako-P0448) secondary antibody. The blot was developed by chemiluminescence Pierce ECL Plus Western blotting substrate systems (Thermofisher Scientific) and immunoreactive bands were detected using the FluorChemE image acquisition system (ProteinSimple) equipped with a cooled CCD camera.
3
Quantifying Synaptotagmin-7 Expression
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Adrenal glands and brain extracts were collected from P0-1 Syt-7 WT and KO mice and lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Invitrogen, 89900). The supernatants were collected and protein concentrations were estimated by using the BCA Protein Assay Kit (Pierce 23227) and plotting the resulting BSA curve. 25 μg and 15 μg of protein, from adrenal glands and brain extracts, respectively, were resolved by 4-12% SDS-PAGE (Invitrogen, Thermo Fisher Scientific) and wet-transferred onto an Amersham Hybond LFP PVDF membrane (GE Healthcare). The membrane was blotted with rabbit polyclonal α-Syt7 (1:500; Synaptic Systems SY105173) and mouse monoclonal α-VCP (1:10000; Abcam ab11433), as a loading control, followed by α-rabbit (1:2000; Invitrogen P0448) and HRP-conjugated α-mouse (1:2000; Invitrogen P0447) secondary antibodies. The blot was developed by chemiluminescence SuperSignal™ West Femto and Pierce™ ECL Plus Western blotting substrate systems (Thermofisher Scientific) and immunoreactive bands were detected using the FluorChemE image acquisition system (ProteinSimple) equipped with a cooled CCD camera.
4
Immunoblot Analysis of Munc13 Proteins
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Extracts from HEK293FT cells expressing ubMunc13-2-EGFP or Munc13-1-EGFP, as well as non-expressing cells, were collected and lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Invitrogen, 89900). The supernatants were collected and protein concentrations were estimated using the BCA Protein Assay Kit (Pierce 23227) after plotting the resulting BSA curve. 25 mg of protein was resolved by 4-12% SDS-PAGE (Invitrogen, Thermo Fisher Sci-entific) and wet-transferred onto an Amersham Hybond LFP PVDF membrane (GE Healthcare). The membrane was blotted with rabbit polyclonal α-GFP (1:300; Synaptic Systems SY132003) and HRP-conjugated mouse monoclonal α-β-Actin-Peroxidase (1:10000; Sigma-Aldrich A3854), as a loading control, followed by HRP-conjugated α-rabbit (1:2000; Agilent Dako-P0448) secondary antibody. The blot was developed by chemiluminescence Pierce ECL Plus Western blotting substrate systems (Thermofisher Scientific) and immunoreactive bands were detected using the FluorChemE image acquisition system (ProteinSimple) equipped with a cooled CCD camera.
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