Bjerrum Schafer-Nielsen buffer, modified by DING LAB, v1.0
Pingtao Ding
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Abstract
This is a modified transfer buffer recipe for a semi-dry Western blot.
Steps
proportion guide
48millimolar (mM)
39millimolar (mM)
15% volume
9.2
This recipe is modified from the original Towbin (1979) buffer, with increased Tris base but reduced glycine.
This recipe is suitable for semi-dry transfer, and ideal for SDS-PAGE and 2-D PAGE.
References:
Garfin DE and Bers G (1989). Basic aspects of protein blotting. In Protein Blotting: Methodology, Research and Diagnostic Applications, B.A. Baldo et al., eds. (Basel, Switzerland: Karger), pp. 5–41.
Towbin H et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350–4354.
for 1L
add in a 2L
58.2g
29.3g
add up to800mL , stir and mix well until all salts are fully dissolved
measure pH, and it should reach around 9.2
top up with ddH2O to 1L
before using, dilute the 10x stock buffer into 1x working buffer
for 1L
take 100mL
add 150mL
top up to 1L
Tips (modified from Bio-Rad: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6211.pdf ):
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Use high-quality, analytical grade reagents to enable better buffer conductivity and hence better transfer.
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If reusing the buffer, measure the pH before use and make sure it maintains at
9.2. -
Do not further dilute the transfer buffer from 1x to the levels below the recommended concentration, because this decreases the buffering capacity of the buffer.
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Do not adjust the pH of the buffer, because it can result in increased buffer conductivity, manifested by higher initial current output and decreased resistance.
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There is no SDS in this recipe, but it can be added when it is necessary. Increasing SDS in the transfer buffer increases protein transfer from the gel but decreases the binding of the protein to the nitrocellulose membrane. In this case, nitrocellulose membrane can be substituted by PVDF membrane when SDS is used in the transfer buffer.
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Addition of SDS increases the relative current, power, and heating during transfer, and may also affect the antigenicity of some proteins.
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Increasing alcohol in the transfer buffer decreases protein transfer from the gel and increases the binding of the protein to the nitrocellulose membrane.
