On roughly a third of my immunoblots, I see these small spots (indicated by red arrows) after I develop the membrane. It doesn't happen every time and I never change my protocol. When it does happen, it appears to be a completely random pattern that never covers the entire membrane. To this point, I haven’t been able to find any information on the cause of this. Some background information on my protocol:
I use the Life XCell SureLock™ Mini-Cell Electrophoresis System for PAGE and transfer. I make my own bis-tris (generally) gels and have observed the spots after transfering 6%, 8%, 10% and 15% resolving phase gels run in TGS, MES, MOPS and tricine buffers. It likely isn't the electrophoresis conditions as the spots do not appear with Coomassie staining. Note: samples I run are mammalian brain fractions and are in a very simple, detergent-free buffer. I then wet transfer for 2 hours at 50V constant (always) in the following buffer:
3.03g Tris / 14.4g glycine / 800ml ddH2O / 200ml methanol
Transfer is onto brand new Bio-Rad nitrocellulose membranes with a pore size of 0.45um. The sponge pads and blotting papers (extra-thick) are also from Life and are brand new. After transfer:
-Rinse blot for 5 min in warm PBS
-Block for 1 hr in Li-Cor Odyssey Blocking Buffer
-Primary antibody incubation in Li-Cor Odyssey Blocking Buffer with 0.15% Tween-20 overnight
-Wash 4x15 min in PBST
-Secondary antibody incubation in Li-Cor Odyssey Blocking Buffer with 0.15% Tween-20/0.01% SDS for 45 min
-Wash 2x15 min in PBST, 1x5 min in PBS
I then immediately develop the blot using the Odyssey Infrared system. The spots seem to be more prominent in the 700nm channel, but that may be because this channel has slightly more background in our lab rending the spots more visible than the 800nm channel.
I have Ponceau stained the membrane after transfer and the spots are not apparent. I have also Ponceau stained a membrane this didn't have protein transferred to it (i.e. transfer an empty gel). I rarely have any bubbles on the membrane so I highly doubt there is any fuzz or anything in between the gel and membrane (this is what I initially though the issue was).
Has anyone observed similar spots on their membranes after developing? It is very frustrating because it doesn't happen every time. Any suggestions would be greatly appreciated.
it is possibly because of ur sponge it happened once for me I pressed too much my sponge and it scratched the filter paper below after that those appeared I just guess ur problem is the same as mine
Dear Grant,
Pre-wet your nitrocellulose in water for the time of gel running. Unfortunately this trick could not help, since your spots seems for me as typical surface damage of the membrane, are lot-dependent. Ask people around for another membrane from different company.
Keep fingers cross - jan
It seems a problem of the mambrane. do you equilibrate the membrane in transfer buffer?
I would say that it is due to the presence of bubbles or either membrane and/or sponge not properly wet before stacking the sandwich.
Thanks for the responses everyone. Some considerations:
Paul - bubbles or spots do not appear upon Ponceau staining of the membrane. I was under the assumption Ponceau was highly sensitive? Also I have not tired another detection method. Will give it a shot.
Sepideh - I am generally pretty careful but will take extra care next time.
Wei - PVDF has a tendency to create background with the Odyssey system. I used to use PVDF and have switched to nitrocellulose for this and a few other reasons.
Valeria - I soak the pads, papers and membranes in transfer buffer at 4degC for about half an hour.
Jan - I will try soaking the membrane for an extra hour next time. Additionally, I will see if anyone in another lab has nitrocellulose from another company.
Jean-Nicolas - The spots do not appear on Ponceau staining. I will soak the sandwich components longer next time.
I believe this a common problem with WB even with a Li-Cor System.
This is unwanted binding of primary/secondary antibody to the membrane.
I would recommend you to be careful with the final washing step (as you have mentioned: Wash 2x15 min in PBST, 1x5 min in PBS). You can re-check the pH of PBS. Good luck.
Jan - my understanding is that nitrocellulose doesn't need to be soaked for long (as I would with PVDF). Will soaking have adverse effects with nitrocellulose?
Niroj - Wouldn't unwanted binding cause greater fluorescence instead of white patches?
Yes these are bubbles. You need to soak your sponges and filters in transfer buffer and use a roller to push all of the air out.
Dear Grant,
With the soaking the nitrocellulose (NC), the rule is should be longer than the minute nowadays (the prolongation of this step would never destroy NC). Ancient NC membranes were soaked for longer time due to the poor quality. Since you did it well, the quality of the membrane is the only suspected... Thank you for your picture added to my collection, the above is welcome as well;)
Best - jan
I have exactly the same problem but already observed with Ponceau staining. Did you resolve this problem?
Regards
Hi Steeve-
Yeah I figured it out. The transfer voltage was a bit too high - it seems the gel got hot enough (even though I run on ice in a cold room) to nearly melt, thus blocking the mobility of the proteins out of the gel. I hadn't considered this possibility initially because there was no 'melted gel' on my membrane. I had to play around with the transfer buffer a bit to figure this out too.
Anyway, reduce the transfer voltage. I was running at 50V for 2hr - I changed to 40V for 3 hrs and haven't had a problem with wet transfer since. If you continue to have issues, switch to a semi-dry system.
Thank you for your answer (and hello --- sorry I forgot it :/)
The best conditions I found out for our samples was over-night at 30V (on ice). It was almost the same results 2h at 100V. 2h with less than 100V shown not optimal proteins transfer for us.
Therefore, I'm surprised it could be a Voltage problem because I don't always have these white splotches. Sometimes everything's perfect, or just one of the two gels! But, I have to admit I've already seen something like a melting gel on my membrane... But just once or twice. And I always check that the A are not to high at the end of the transfer (always between 50-70mA for over-night condition) and Ice is still be present.
So, I really consider the idea it could be a Voltage problem, but do you think I could go lower than 30V over-night? And I'm surprised that I don't remember to have already seen this kind of melting gel with our past conditions at 100V, the only differences are we are using 1.5mm gel instead of 0.75mm and we are using nitrocellulose instead of PVDF (because of Odyssey...) I'm trying right now with our old sponges as I read somewhere it could be the system is too much tight...
(Hope my english is understandable)
Thank you once again for your opinion
Best
In my opinion, you could go lower than 30V. I am actually in the process of standardizing transfer of high molecular weight proteins, and have seen people go as low as 15V overnight for this.
I bought new sponges when I was having this problem and that didn't resolve anything, but it may help in your case. Are you using fresh transfer buffer (I assume Towbin?) each time? Making new buffer just before transfer seemed to help me out a bit. As far as gel thickness, proteins will more readily transfer out of thinner gels (in general, sometimes bigger proteins require the space in 1.5mm gels to readily mobilize). Also, with your thicker gel, you may actually be making the sandwich "too tight".
But as far as the splotching, I would try a lower voltage. If you can't, maybe reduce the methanol in your buffer. Though methanol helps immobilize proteins on your membrane, it can reduce mobility of proteins out of the gel. Let me know if this works for you.
Using Bio-Rad "stain-free" TGX midi gels (or standard Bis-Tris) I get these same transferless "splotches" onto Amersham Protran Premium nitrocellulose using cold room 20V overnight wet transfers. I've tried fast and slow transfers and it's made no difference. Using the LICOR, I can preimage the blot before immuno and --since the ladders are prestained-- can see the splotches before spending time and material on immuno if the splotches are in the band region of interest. To see them, I use ImageJ (FIJI) and view the 700nm channel with Image/Adjust/Brightness and Contrast set to near maxium values. Prewetting and presoaking variations have no effect. Duration and voltage of transfer have no effect. I've also had them using Bio-Rad PVDF and using ECF for display. Changing membranes in that case had the most effect but not a complete consistent solution. These are definitely not bubbles. Would love a solution for this one. Grant, have you refined your method to resolve this once and for all?
It's been a long time since we discuss about this problem but I guess i solved this issue (or at least reduced it enough) and I think this still could help some people... After transfer, simply try to "unstick" your gel from the membrane as gently as possible. This worked for me. Maybe some proteins were taken off from the membrane when the gel is sticking a bit. Good luck!
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