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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Tris and transfer buffer
Posted by: BioStudent (IP Hidden, New member, 1)
Date: April 26, 2005 02:30PM
Hi,
Can anyone telle me exactly: 1. What transfer buffer does to the blotting pads and the PVDF membrane and Why transfer buffer? What exactly is so sepcial about it? 2. What exactly happens to the membrane when being blocked in Tris buffer? Links would be great :o) I'm sorry if I come across a bit ignorant. My excuse: I'm a first year student :o) Thanks guys BioStudent
Re: Tris and transfer buffer
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: April 27, 2005 12:48PM
First in order to add electricity to the system, some liquid buffer must be used to carry the current. During transfer the gel sandwich is assembled in such a way that the proteins will be transferred from the gel itself to a membrane (PVDF if you like). The electrical current forces the proteins out of the gel and onto the membrane. The components of the transfer buffer help the protein to migrate from the gel and adhere to the membrane. Methanol for instance can improve binding of SDS proteins to nitrocellulose membranes. Elimination of methanol results in increased transfer efficiency, but diminishes binding to nitrocellulose membranes. Thus with a nitrocellulose membrane, it is important that the transfer buffer contain methanol. Use of PVDF membrane for SDS protein transfers eliminates the methanol requirement and will allow more efficient transfer of high molecular weight proteins or difficult to transfer proteins. This is because methanol causes gel pores to contract which results in fixation of large molecular weights within the gel matrix. Keep in mind that PVDF must be prewetted in 100% methanol before use, or nothing will bind to the membrane. Addition of SDS detergent to the transfer buffer has been reported to increase transfer efficiency (we use it ourselves, otherwise the transfer is poor). SDS aids in eluting the proteins from the gel, but may reduce binding to a nitrocellulose membrane. SDS also increases relative current, power, and heating so you may wish to transfer at lower power in the cold. SDS may also affect the antigenicity of some proteins, which rarely can be a problem in specific protein detection through antibody application. The blotting pads themselves- sponges- merely help keep the gel from drying out and allow the passage of the transfer buffer bearing the proteins thorough them. The same thing happens with the filter paper. Please forgive me if this explanation is too long. I hope it helps. :) I have to go now, but will answer your other questions shortly.
Re: Tris and transfer buffer
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: April 27, 2005 12:55PM
Almost forget, you asked for links. To review what I have just told you about buffer formulation, check out these sources:
Gershoni, J.M. and Palade, G.E., Anal. Biochem. 124, 396 (1982). Gershoni, J.M. and Palade, G.E., Anal. Biochem. 131, 1, (1983). Towbin, H., Staelin, T, and Gordon, J. Proc. Nat. Acad. Sci. 76, 4350 (1970). Towbin actually developed the Tris/glycine/Sds buffer. It is also known as a towbin buffer. while in the tris blocking buffer, non-specific sites are blocked on the membrane. this helps to ensure that antibody binding is specific.
Re: Tris and transfer buffer
Posted by: Xu Lin (IP Hidden, Unregistered user, )
Date: June 20, 2005 01:56PM
do you think it is neccessary or helpful to pre-wet the Nc membrane in 100% methanol before use
Re: Tris and transfer buffer
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 20, 2005 04:20PM
It is not neccessary to prewet Nc in 100% methanol. We get ours from Bio-rad and according to their instructions we prewet the membrane in distilled water and then equilibrate the membrane in transfer buffer for at least 10 minutes before use. I have never prewetted in methanol, so I cannot tell you whether it would be helpful or harmful. Only that it is not neccessary.
Re: Tris and transfer buffer
Posted by: john h (IP Hidden, Unregistered user, )
Date: June 20, 2005 05:36PM
Xu Lin Wrote:
------------------------------------------------------- > do you think it is neccessary or helpful to > pre-wet the Nc membrane in 100% methanol before > use > > Only PVDF needs to be activated using methanol before blotting.
Re: Tris and transfer buffer
Posted by: shane (IP Hidden, Junior member, 10)
Date: August 12, 2005 11:18AM
Biological science is all about buffers, and ur buffers change as ur pH changes. For acidic pH u normally prefer the acetate and the citrate buffers, for physiological pH u generally do with phosphate buffer and at pH 8.3 to 8.6 u use tris buffer. WHY?????
A buffer of that pH is used that has a high buffering capacity at that pH, since transfers is at 8.3 and 8.6, tris has a high buffering capacity at that range. But is does not mean that others dont work. u would be glad to know that proteins are coated to elisa plate at pH 9.6 and its carbonate buffer and the buffer system is different. now blocking the membrane after transfer can be done with phosphate or TRIS the end result is the same as the membrane has affinity to proteins. i hope i have provided some justification and clarification. urs shane
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