Antibody too much? - Signal not consistent, and fades quickly (Dec/01/2010 )

Hello all,

This is a summary of the most recent western I have run:
- Blocking: 5% nonfat milk in PBS-T (0.5% tween); overnight @ 4deg
- Brief rinse: 2min/RT
- Primary (mouse monoclonal)- for 4h/RT in PBST. Dilutions tested 1:1000, 1:2000, 1:3000
- PBST rinses
- Secondary (goat anti-mouse)- 2h/RT in PBST. Dilutions tested 1:4000, 1:5000
- PBST rinses
- ECL: 5min
- exposure to film: 5 min

When I tested 1:1000/1:4000 combo of the two antibodies, I got a terrific signal with 5 min exposure, but the signal faded soon after. Upon calling the GE tech support, I was told it was because my antibodies were too high. So I diluted them both some. I kept the secondary at 1:5000, and compared the primary at 1:2000 and 1:3000. The latter produced results, but not as good as the very first one (1:1k/1:4k dilutions). However, I am still noticing the same problem of signal fading after the initial 5 min exposure.

I should also mention that these results are with recombinant protein (positive control). With cell lysates (where it really matters!), I loaded 10ug and 20ug of total protein, and did not see any bands at all (with 1:2k/1:5k; and 1:3k/1:5k)

Any suggestions?

Thank you in advance for your help.

Can you please explain, how the signal faded. Did you take another piece of film and exposed again for 5 min and observed a lower signal (no signal at all???)? Which ECL substrate and film are you using? Which primary and secondary Abs are you using? The dilutions are very different for different suppliers, so it's really difficult to say anything based on just the dilutions. Did you see any white spots inside your black spots (a.k.a. ghost bands)?
As far as the cell lysates go, how long did you expose the film? Did you make a fresh batch of developer? How long did you develop? Are you sure your protein is expressed? You can always load even more cell lysate and see if you get anything. You can expose up to about 60 min. And be sure to use a fresh developing solution - it really makes a big difference. And if you stop developing when you see the band for your positive control = the rec protein, dilute the positive control. There are a number of suppliers of different substrates and films that enable very low detection levels.

Best of luck.
Miha

Thank you for replying, Miha.
Yes, I did lay down a second film after the first 5-min exposure and that came up blank.

As far as your question about suppliers: secondary antibody and ECL are both from GE. Primary is from santa cruz. Film is Kodak BioMax MR.

I did not see any ghost bands on any of the films.

Film exposure for cell lysates was for the same duration as the positive control (they were on the same blot):5 min

As for the developer- we use a shared dept. resource. It is Kodak X-OMAT processor, where the developer and fixer are added to the machine as needed. Since I am not doing manual development, using fresh developer exactly isn't quite possible.

What would you suggest?
Do you need additional information for my expt. settings?

Thanks a miilion!
- Reeteka

Ok, here are my thoughts.
We have had bad experience with Santa Cruz antibodies, so we generally try to avoid them. However, if you say that your positive control stains normally, then the primary Abs should be OK.
The substrate and film, I have tried before and work OK, not the best though. I use Pierce SuperSignal West Pico and Pierce CL-XPosure Film. The secondary I have never used GE, we use Sigma or Jackson ImmunoResearch.
I am not sure why your signal fades so quickly, I am quite sure it is not because the concentration of Abs is too high. I would suggest loading more protein onto your gel, until you observe saturation or ghost bands. The same for your cell lysates.
Good luck.
Miha

Hi Miha,

I took your suggestion and loaded more protein - with cell lysate samples, I loaded 60ug/lane. I should mention that the sources of cell lysates are two different cell lines. And on analyzing both now, it seems that there is significant difference in basal expression levels of my protein of interest in these two lines. I say so because one of the lysates showed bands upon loading 60ug total protein, although they were not as crisp as I'd like to see. They were bit fuzzy, with some smearing between the bands (the protein has different isoforms, so shows up as three bands - that's expected)
In the second cell line, I saw ghost bands upon loading same amount of total protein - and that's why I think that these cells express more of my protein than the first cell line. In fact, this blot had some lanes that were glowing in the dark! For this problem, I think I'll strip the blot and re-probe. Will definitely dilute the secondary more, do you think I should dilute the primary as well?

A more worrisome issue is that the positive control looks different in the two blots, even though I used the same dilutions of antibodies (1:4k primary, and 1:10k secondary) in both blots. The difference is that the blot yesterday showed nice,crisp bands in the positive control, even though that wasn't the case with the cell lysate, as I explained above. And the blot I developed today (with same amount of protein loaded, and same dilutions) the positive control is a total blur! No bands can be seen. Do you have any thoughts what would cause this difference?

Thank you again for your input. I really appreciate your help in this matter.

Sincerely
Reeteka

Reeteka,

I generally keep my primary and secondary Ab dilutions pretty much constant. In my experience the amount of protein loaded on the gel (and the condition of the sample) is the determining factor whether a WB is nice and crisp or not. If you load a lot of protein the bands will glow in the dark and leave a brown stain on the membrane. That's normal. If you get ghost bands, you should load less protein. Otherwise I would not meddle to much with the Ab dilutions, because if you change too many parameters at once, you will not know, which is responsible for your results.
For your positive control, as well, I suppose it is the cause of the sample, not the Abs. However, there is little I can say without seeing the blot. Was the sample old, did you add a lot of 2-mercaptoethanol, do you see anything (for example a dot instead of a band), etc...?
Miha