Hi! There is a limitation on this technique, which is the distance between residues available for the cross-linking. Eventually, the crosslink between your monomers would not occur, although this is normally expected to happen, since there are many reactive aminoacids on the protein's surface. One thing that might influence your result but is usually neglected is the reaction pH, which will affect the ionization state and reactivity  of some of these aminoacids, and also of the protein as a whole, leading to different conformations and reactivities. Perhaps you already tried different pHs for your reaction? In our hands, between pH 6.5 and 8.5, the more alkaline the pH, the faster the (internal cross-link) reaction occurred. We also had problems with an old glutaraldehyde solution, which probably was degraded and was poorly efficient for cross-linking. however, you are using high glutaraldehyde concentrations, therefore cross-linking would, again,be expected. When you say you don't get a dimer, do you see high molecular weight aggregates on top of the SDS-PAGE? How pure is your protein preparation? Maybe your protein  forms high molecular weight aggregates and runs on the top, or it cross-links and aggregates with other proteins in your prep? Let me know if I can be of any further help. I also append three papers (one paper and two links) that could help you to find better conditions for your reaction. Best regards, and good luck for you!

https://www.google.com.br/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&cad=rja&uact=8&ved=0CDsQFjAFahUKEwihp9mZmqTHAhWFgpAKHbJBB44&url=http%3A%2F%2Fwww.fgsc.net%2Fneurosporaprotocols%2FHow%2520to%2520cross-link%2520proteins.pdf&ei=iJTLVaHDOIWFwgSyg53wCA&usg=AFQjCNEOb9d33uxY3WkJ22gW6-ln8t0ukg&bvm=bv.99804247,d.Y2I

http://www.sciencedirect.com/science/article/pii/S0003269707007026

Article Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-...

Dear Julio Alberto Mignaco ,

Thank you so much for your valuable suggestion. I tried with two different pH of 7.5 and 7.8 (which I used to elute my his tagged protein) and on the other hand yes! I am getting higher molecular weight  bandwhich is almost near the well in 7% SDS PAGE also. 

In some gels I am getting a very light smear type band. what does it mean?

Thanks for your links and let me try your suggestions and the suggestions provided in the links.

Hi again! If you get hi-weight aggregates, possibly your protein is being trapped on them, as a consequence of extensive cross-linking. Perhaps diminishing the glutaraldehyde concentration might help. You see that in our experiments we used something between 0.15 and 0.3 mM glutaraldehyde final, which is roughly on the 1/1000 range of the glutaraldehyde you are using. If you have time, read the papers on the links (hope you can access the pdf's) to get more hints.  I would also suggest you try a time-course experiment using much less glutaraldehyde to make the reaction, on the low millimolar range. But seek a bit more information to confirm whether this is a good suggestion, before you spend more time and protein on a wrong experimental condition. Once again, best regards and good luck for you!

Hello. You could try a range of glutaraldehyde % and a time-course experiment. As a start, you can try using a final concentration of 0.0025% and 0.025% of glutaraldehyde in your reaction. For each reaction, you can take samples at t=0,5,10,15 and 30 minutes. Depending on you result, you can narrow down the % of glutaraldehyde in your reaction to look for the optimum % needed. . ie: 0.005%, 0.01% or something like that. It is advisable to use lower % of glutaraldehyde in your reaction to observe the formation of lower oligomeric complexes.

Thank you Ju Lin Tan. I will try your suggestions.

Try 1-50 micromolar zero-length copper-phenanthroline cross-linker

Hi Vijaykymar, you could try using lower concentrations of glutaraldehyde. Make a stock of 5% and start using 0.05 % of total glutaraldehyde. Simultaneously, 0.1, 0.25, 0.5 and upto 1% can be used. I do this very often. After adding glutaraldehyde to your sample, incubate it for 2-5 minutes at RT or 37C, then use SDS loading buffer and boil it for 10minutes.

Dear colleagues: For those who are still interested in using glutaraldehyde as protein cross-linker, I found an excellent review paper published in 2004 and online earlier in 2018. It will certainly be helpful to you. As it is an open access publication, I hope the authors will not get angry with me because I posted its link... Best wishes to all!

Article Glutaraldehyde: behavior in aqueous solution, reaction with ...

Dear Julio Alberto Mignaco,

Thanks a lot for your answer. I am interested in using glutaraldehyde as protein cross-linker and your expertise helps me a lot.

Best regards to you and to all!

Julio, muchas gracias!!!!

I worked on this back last century. Search for Ross, McIntosh, crosslinking, Ca-ATPase, sarcoplasmic reticulum, glutaraldehyde, monomer, dimer, active site...

Thank you so much David Ross!

You're welcome. Did you find the paper that shows that glutaraldehyde forms the active site crosslink between a lysine and an arginine?

Yes, I found it. Thank you David Ross!

Hi, I used glutaraldehyde at a final concentration of 0.09% for 15 minutes on ice, followed by western blot analysis for p53 dimerization and tetramerization (JCS, 2010).

Best

Jian

• Hi Vijaykymar, I'm trying to do cell cross linking assay for my project recently, unfortunately, I failed many times. Can you give me some advise?
• My target protein is PKM2 whose monomer is ~58Kda. I tried to crosslink the protein with 0.5%, 2.0% of glutaraldehyde with various durations from 3 minutes to 15 minutes. But none of the methods I tried help me to get the perfect bands. Can you please provide your valuable suggestions and expertise.?

Glutaraldehyde blurs the protein bands and causes the protein to run slightly faster on a gel. I'm 6 years late, but I see possible dimers in Vijayakumar Rajendran's gel (middle lane). You can visualize proteins with negative stain transmission electron microscopy.