My group have nicely established glutardehyde cross-link to study the complex formation of the protein of our interest.We are working on both total tissue/cell extracts or recombinant proteins, highly pre-purified by gel-filtration columns equilibrated with PBS or Hepes. To verify our results we are at the beginning of the fight with set-up of the BS3 cross-link. All our attempts failed at this moment, the proteins or precipitate, or migrates as an smear, or no cross-link is observed. Simple reading of the protocols or literature search gives us impression this chemical is working everywhere apart of our lab. Please share with us any experiences, especially these are so obvious, no one is describing it...
Hi Jan, the BS3 crosslinking reagent has a much longer spacer arm than glutaraldehyde.
As such, you will get crosslinking between distant proteins. If you treat a concentrated protein solution with BS3, you will get non-specific crosslinking with every protein within ~1 nm.
As for the failed crosslinking reactions, the NHS esters on BS3 are very labile. They have to be dissolved and stored under strict anhydrous conditions.
I asked about how to store HNS esters on RG and got the answer that you have to do it in a specific way. Otherwise you will lose NHS reactivity:
https://www.researchgate.net/post/Storing_N-hydroxysuccinimide_NHS_esters_without_losing_reactivity2
Hi Jan, the BS3 crosslinking reagent has a much longer spacer arm than glutaraldehyde.
As such, you will get crosslinking between distant proteins. If you treat a concentrated protein solution with BS3, you will get non-specific crosslinking with every protein within ~1 nm.
As for the failed crosslinking reactions, the NHS esters on BS3 are very labile. They have to be dissolved and stored under strict anhydrous conditions.
I asked about how to store HNS esters on RG and got the answer that you have to do it in a specific way. Otherwise you will lose NHS reactivity:
https://www.researchgate.net/post/Storing_N-hydroxysuccinimide_NHS_esters_without_losing_reactivity2
I found that the concentration of BS3 made a huge difference when crosslinking a dimer. See this article (http://www.ncbi.nlm.nih.gov/pubmed/25003324). At a high concentration, such as 1 mM, the protein bands were very broad. I interpreted this as being due to multiple molecules of BS3 reacting with each protein molecule. Reducing the concentration to 10 micromolar resulted in a tight dimer band.
As you got smears, it looks like you used more than required cross-linker. Use minimum concentration of cross-linker.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques