Forum :: Inverted curve - continued


Welcome, Guest
Username: Password: Remember me
These are the posts of the old forum. It was not possible to transfer the user data, so they are missing in most of the posts. For new questions, go to the general discussions.

TOPIC: Inverted curve - continued

Inverted curve - continued 29 Aug 2011 02:00 #1

I did a study of the following buffers with the following NaCL concentrations - 0.15, 0.25, 0.35, 0.5M. 0.15M is what I typically use. Seems like after 0.15M it starts an inverted peak. Top one is 0.15, then 0.25, 0.35, and bottom is 0.5M. Does not make sense since back in 6-15, it worked...
mtarca


5.jpg

Inverted curve - continued 29 Aug 2011 02:00 #2

I see you use automatic reference subtraction. How are the raw curves? 0.5 M should give you a huge upwards jump.
Now you find -50 RU (0.5 M NaCl) after reference subtraction. It could be that the two channels react differently to the salt concentration due to different surface immobilization. It seems to me this is the case here, because the effect is concentration dependend.

Changing the FC. hmmm, Is system check alright? Is buffer injection allright? (pipet directly out the buffer bottle)--> there should be no differences in channels and after reference subtraction.

Arnoud

Inverted curve - continued 29 Aug 2011 02:00 #3

Top curve is from 0.15M. Bottom is from 0.5M solution. It seems like for the 0.15M I do see an increase. But not that much for the 0.5M NaCL. I am sure I got it from the 0.5M NaCL bottle.

6.jpg


7.jpg

Inverted curve - continued 29 Aug 2011 02:00 #4

I am still a bit confused about which curve you subtract from which curve. If you inject the 0.5 M NaCl solution do you record two curves (1= with ligand and 2 = reference)?

Looking at curve 6 and 7 I should say this are not raw data since the buffer jump should be different between 0.5 and 0.15 M. Do you change the running buffer also to 0.15 and 0.5 M?

Do you inject salt only (no analyte)? Then you should not expect a binding curve but a buffer jump and flat line. You can use this curve for compensating differences in channel responses (double referencing).

The other thing is that running in 0.5 M NaCl will diminish binding of a lot of proteins. In fact is is a know recipe to dissociate proteins from sensor chips or columns.

Still confused whats going on.

Arnoud

Inverted curve - continued 29 Aug 2011 02:00 #5

Sorry for the confusion. I did another simple experiment. Using FC 2, I injected the following: 1) 0.15M NaCL buffer only, 2) TSH in 0.15M NaCL, 3) 0.5M NaCL buffer only, and 4) TSH in 0.5M NaCL buffer. Each sample having each own cycle. I started with the 0.15M buffer and switched to the 0.5M buffer. The sensorgram below are labeled. Maybe try a diff. chip? running out of ideas...

8.jpg


9.jpg

Inverted curve - continued 29 Aug 2011 02:00 #6

That is funny, you should expect with 0.5 M the same as with 0.15 M. I am also out of options why this happens and how to solve.
Try another chip or an other ligand analyte pair to make sure the machine is working properly.