Hey,
I have a question regarding the stain free blot technology of Biorad. After electroforesis I take a picture ("Stain-free gel") for activation of the trihalo-compound. Next I transfer the gel to the membrane using TransBlot Turbo mini TGX (3 min transfer) and whash it 3x 5min in TBST. Next, I take another picture using the Chemidoc (Stain-free blot). This picture is very important as it is used for normalization (all protein normalization). Next, I execute the rest of the protocol and take other pictures (background= very low). However, the background on the Stain-free Blot image is very high and interferes with my normalization, making it unreliable...
I have increases whashing steps (another 2x 5min), Tween concentration,...
Does anybody have a suggestion how to overcome background issues?
Hey Caroline,
I think the manufacturer's protocol images the membrane directly after blotting with the Trans Blot Turbo Pack. You could try to skip the washing steps with TBST.
As far as I know, Tween can contribute to the background, so I rinse my blots in TBS before detection (although I use the classical workflow/ wet blot as we do not have the TransBlot Turbo system).
The buffer system is proprietary, so maybe there is no Tween in the blotting buffer of the Trans Blot Package.
Best,
Marianne
Agree with Marianne: you should image your gel directly after running it. I don't know to what extent the halo-protein complex is stable post-transfer and your "background" problem might be a "signal" problem. In that sense the methodology is not ideal for normalization since comparisons are made between pre and post transfer signals (which might differ due to transfer variability), rather that post-transfer signals only. To address this, you could try various post-transfer reversible fluorescent stains but tread with care: reversibility is not always complete and might give you background fluorescence for fluorescence based immunodetections.
Hey Marianne Musinszki en Sébastien Soubeyrand ,
Thank you for your response!
In the past, I have used TBS instead of TBST, however my results were better using TBST? Furthermore I have found a protocol of Biorad that takes a picture prior to adding ECL substrates for the normalization steps. (see Bulletin 6390).
However, I washed this a million times and still I had a lot of background/signal noise... I have taken multiple pictures and I saw that the noise seems to be depending on the humidity of my blot? Real wet/ dry blots have a lot of noise whereas semi-dry blots have less noise (however, then airbubbles are interfering with my pictures) and I believe I have still to many noise for normalization?
My chemiluminscence and colorimetric blot seems fine?
Any suggestions?
Hi Caroline,
I assume that you are using a Nitrocellulose blotting membrane. If you switch to low-fluorescence PVDF membranes, you will get almost no background.
Best regards,
Toni
Hi Anton Posch ,
No, I have used PVDF membrane (soaked in ethanol until it becomes translucent (+/- 2 min) and next in the transferbuffer). For the transfer, I use 14 Transblot Turbo Mini Size transfer Stacks (2stacks of 7; up and down)) and use the mini transblot turbo TGX setting (3 min)?
Kind regards,
Caroline
Hello Caroline Merckx ,
unfortunately I can't identify the problem... but there are some points I can mention, maybe this helps you optimizing the procedure.
When using PVDF mebrane, be sure to use a low fluorescent type. I activate the membranes 90 seconds in methanol, not ethanol. Then wash it really good in transfer buffer for 15-20 minutes. It does not really become translucent, it is more like white-glossy. However, your cited technical bulletin uses a nitrocellulose membrane for the V3 workflow...
Do not let the membrane dry out once you started activation. You can see the color changing to plain white if it gets dry. For all detection steps, try to red rid of most of the buffer by holding the membrane edge onto a tissue, but do not let it dry - you have to be quite quick, do not let it sit in air long, for example when assembling the transfer pack or in the imager. Some collegues of mine put the membrane between pieces of plastic wrapping film (also for ECL detection), but I don't know how this would impact the signal of a stainfree blot.
As you already posted, the membrane of SFblotdrypriorECLsubstrates.jpg seems too dry. However, the big black spots look more like air bubbles during the transfer to me. Be sure to roll out air from the assembled transfer pack and have a contimuous buffer film to ensure electrical contact on the whole membrane area.
I have no experience with any Turbo blot system, but judging from the background pattern, the whole thing may be a bit too dry.
To clarify the TBS advice: For the washing steps I also use TBST to remove nonspecific bound antibodies, for example 3x15 minutes. Only after that I additionally rinse it in TBS to remove the Tween.
(The technical bulletin places the membrane in water before imaging the stain free blot).
Good luck still,
Marianne
http://www.bio-rad.com/en-us/product/immun-blot-low-fluorescence-pvdf-membrane?ID=LN7REQ15
Hi Marianne Musinszki and Anton Posch ,
Thank you for your answers! Apparently we are using normal PVDF membranes and not low fluorescent PVDF membranes...
I really hope this low fluorescent PVDF membrane will make a difference
Thank you!!
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