Treated or infected cells were washed twice with PBS. Whole cell lysates were collected in SDS-PAGE loading buffer containing 4 M Urea (Sigma-Aldrich, U0631) and 50 mM Dithiothreitol (DTT; Sigma-Aldrich, D0632). Proteins were resolved on NuPAGE 4–12% Bis-Tris Protein gels (Invitrogen, NP0322BOX) in MES (Invitrogen; NP0002) or MOPS buffer (Invitrogen, NP0001) and transferred onto 0.2 μm nitrocellulose membrane (Amersham, 15249794) for 90 min at 30 volts in Novex transfer buffer (Invitrogen, NP0006-1) according to the manufacturer’s instructions. Membranes were blocked in PBS with 5% FBS (Block) for a minimum of 1 h at room temperature. Membranes were incubated in primary antibody diluted in Block for a minimum of 1 h, washed three times with PBST for 5 min each, then incubated in secondary antibody diluted in Block for 1 h. Following three 5 min washes in PBST, one 5 min wash in PBS, and one rinse in Milli-Q H2O, membranes were imaged on an Odyssey Infrared Imager (LiCor). The intensity of protein bands was quantified with Odyssey Image Studio Software.
Novex transfer buffer
Manufactured by Thermo Fisher Scientific
Novex transfer buffer is a laboratory solution used in the transfer of proteins from electrophoresis gels to membranes for further analysis, such as Western blotting. It is designed to facilitate the efficient and consistent transfer of proteins during the blotting process.
Automatically generated - may contain errors
2 protocols using novex transfer buffer
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Protein Analysis Protocol
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Treated or infected cells were washed twice with PBS. Whole cell lysates were collected in 1x SDS-PAGE loading buffer containing 2.5 M Urea (Sigma-Aldrich, U0631) and 150 mM Dithiothreitol (DTT; Sigma-Aldrich, D0632). Proteins were resolved on NuPAGE 4–12% Bis-Tris Protein gels (Invitrogen, NP0322BOX) in MES (Invitrogen; NP0002) or MOPS buffer (Invitrogen, NP0001) and transferred onto 0.2 μm nitrocellulose membrane (Amersham, 15249794) for 90 min at 30 volts in Novex transfer buffer (Invitrogen, NP0006-1) according to the manufacturer’s instructions. Membranes were blocked in PBS with 5% FBS (Block) for a minimum of 1 h at room temperature. Membranes were incubated in primary antibody diluted in Block for a minimum of 1 h, washed three times with PBST for 5 min each, then incubated in secondary antibody diluted in Block for 1 h. Following three 5 min washes in PBST, one 5 min wash in PBS, and one rinse in Milli-Q H2O, membranes were imaged on an Odyssey Infrared Imager (LiCor). The intensity of protein bands was quantified with Odyssey Image Studio Software.
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