Recently I have been doing western blot to detect TIGIT, a membrane molecule which usually forms dimers on immune cell membranes. The blots are very clear, but their positions do not match the molar weight of TIGIT: they locate at around 50kD, adjacent to the control protein beta-tubulin (~50kD), while the MW of TIGIT is 26kD.
My first idea is that the TIGIT that is detected is in the form of dimers. So I renewed my sample buffer (450ul 4x Laemmli sample buffer, 50ul beta-mercaptoethanol), and tried to heat for a longer time (from 10min to 20min, the heating temperature is 97 degrees in Celsius), which was in vain.
The total protein amount for each sample is 20ug.
PS: The image of the WB is attached, which is acquired via LI-COR. Red bands TIGIT, and Green bands are mainly beta-tubulin. Another issue is that there is also a clear band of "half" beta-tubulin blots which locate at the 25kD site.
Update: I tried to maintain the sample at 25 degrees or heat it at 60 degrees for 20 min, since a answer in another question of researchgate mentions that heating may lead to stronger hydrophobic effect of membrane proteins to lead to formation of dimers, here is the result: strong bands at 90kD, 50kD, 38kD and 30-38kD.
Update: I tried to add 50mM DTT in addition to beta-mercaptoethanol, heating for 20 minutes at 80 degrees. Now the band on 50kD disappear, but there is no significant increase in 30-38kD bands. There is no band at 20-25kD for TIGIT.
(red: beta-tubulin, control. green: TIGIT)
The WB looks really good. The TIGIT in SDS-PAGE gives a Mr of approx 50 kDa and seems to exists as a stable dimer even after heating and reducing step. Also add 1% SDS to sample buffer and rerun the PAGE
I'd never work with such molecule, however I worked with a viral capsid protein wich native state is as a awefully stable 60 units homopolymer. We got rid of multimers by adding DTT to x50 Laemmli sample buffer plus B-mercaptoethanol. Good luck.
Try whit a gradient gel maked whit more acrilamide for example in an interval of 10 to 20% see my image, I maked the gels in an inteval 4-20% and I did see proteins of 14kDa.
As you see my english isn't good but if you speak spanish I'll tel you how to make them
- Badges
- Science method