It's funny... because people seem to have made quite contradictory experiences... some suggest to avoid milk... others report about less success with BSA. Then there is the question of nitrocellulose vs. PVDF... in our hands e.g. there was a problem with high background when using the combination of PVDF and BSA, while NC and BSA gave much better results.

In the end it is trial and error I guess... depending on your membrane supplier, the antibody you use, etc. But from the variety of experiences it seems to be worth to invest the extra money for the LI-COR BB.

Btw.: Most people using the LI-COR probably know this already... never ever use a standard roller pen to mark the membrane (like you probably used to do in the old days when using X-ray film). The ink will smear across the membrane and give the hell of a background (even if you don't see the ink by eye... but the sensitive IR-LASER of the machine will detect any hint of blue as strong signal).

I have never really had much problem using milk/tween; fish gel; Odyssey blocking buffer; or BSA in terms of background problems with the Odyssey.

We get good results from LiCor's own blocking buffers for the Odyssey system - eg https://licor.secure.force.com/catalog/LI_ProductDetailsPage?sku=927-40000&viewState=DetailView&store=bio&parentCategory=a0d60000000T9QPAA0&navigationStr=ListProduct&searchText=

See one of our recent papers: https://licor.secure.force.com/catalog/LI_ProductDetailsPage?sku=927-40000&viewState=DetailView&store=bio&parentCategory=a0d60000000T9QPAA0&navigationStr=ListProduct&searchText=

good luck.

We use Odyssey Blocking Buffer which works very well. You can use other buffers too, however, you just do not want to use milk or Tween added to your buffer until after the blocking step.

We have tried two different blocking buffer ("Odyssey blocking buffer" (Li-Cor), "Blocking buffer for fluorescent western blotting (Rockland)") and it seems that both work well. Good Luck!

We too use the Odyssey blocking buffer- but I've also used Millipore's Blok-FL- with the SNAP-ID with good success.

the Licor blocking buffer is the best

I used my regular blocking buffer (5% milk with 0.1% Tween-20 in 1x TBS) and it works great

The Odyssey Block works well, but we cut it 1:2 with TBS as it is fairly stringent. As Tumay suggests, for sure don't add detergent to your block!

I've gotten great results with 5% milk in PBS-tween. I've also used the blocking buffer they sell, and had good results as well. But the milk is cheaper so that's what we use.

We have never had issues with 5% milk in TBS-T.

For blocking 5% milk is great, but don't add any tween to the blocking agent. When doing antibody dilutions you can put the tween back in.

Blocking buffer from Odyssey diluted with TBS (vol to vol) work great. Also never use tween before blocking. after transfert and just before blocking you can rinse with TBS (no Tween).

Skimmed milk or BSA works well as the blocking reagent. It never gave problem to me. You can use any of them as well as can try some of the above.

Best Regards

We use 5% BSA in TBS-Tween. We've had no problems with background, as long as we switch to PBS or TBS before imaging. In our experience, the Li-Cor blocking buffer creates a speckly background.

Do you control that the membrane is the good one for Li-Cor. Because, the major background problem come from membrane. You need to choose the good one, compatible with the infra-red of the licor.

Immobilon-FL and Li-COR blocking buffer always worked well for me. Many primary antibodies react with milk proteins, so I would always avoid milk even when not working with the Odyssey :)

I had the background problem when I used milk instead of Li-COR blocking buffer. 5% BSA in TBS resolved the issue for us, please make sure you use RIA grade BSA (it is very expensive). You can reuse BSA for several times if stored properly at 4 degrees after each use.

Hi Denis. We use the Immobilon PVDF-FL membranes and we block in 5% skim milk in TBS, or 3% BSA in TBS. The odyssey blocking buffer works well but is expensive!

It is better to use the Odessey blocking solution which is highly optimized to reduce the background in comparison to milk or BSA. Another thing is to keep in mind that the scanning of dry blot will reduce the background levels greatly.

Good luck..!!

We usually use 5% Mild in PBS or TBS to block, since the blocking buffer Odyssey blocking buffer is quite expensive. This one we use only when we don't get the background under control.

sorry, I pasted in the wrong link for the paper I mentioned, see here:

http://www.sciencedirect.com/science/article/pii/S0303720713002542

Use BB Licor Buffer and pvdf millipore membrane to reduce background. 5% BSA is not good for Licor machine. Be careful to the dilution for the II antibody because they have very strong signal. The dilution for the II ab is very important. Usually II ab dilution for actin or tubulin is 1:8000

One way to reduce background is to reduce the concentration of the secondary antibody. We have use 1:50,000 dilutions of the LiCor secondaries with excellent results depending on the primary antibody. We always use the LiCor buffer, most of the time in a 1:2 dilution. The technical service in LiCor is very good in helping solve your specific problem. You can call them or email them and you will get an answer from people that have a lot of experience and have collected the experience of a lot of user.

This does sound like a blocking issue. I've had good luck with LI-COR BB, some success with milk, and generally less success with BSA. Most blockers are compatible with the Odyssey, so I'd suggest you try whatever you have in the lab and see what works best. To get an answer quickly you can run many lanes of your sample, cut the blot into strips, and process each strip with a different blocker. Then reassemble the blot and scan. I attached an application note that might be useful.

http://biosupport.licor.com/docs/GoodWesternsGoneBad_11169.pdf

It's funny... because people seem to have made quite contradictory experiences... some suggest to avoid milk... others report about less success with BSA. Then there is the question of nitrocellulose vs. PVDF... in our hands e.g. there was a problem with high background when using the combination of PVDF and BSA, while NC and BSA gave much better results.

In the end it is trial and error I guess... depending on your membrane supplier, the antibody you use, etc. But from the variety of experiences it seems to be worth to invest the extra money for the LI-COR BB.

Btw.: Most people using the LI-COR probably know this already... never ever use a standard roller pen to mark the membrane (like you probably used to do in the old days when using X-ray film). The ink will smear across the membrane and give the hell of a background (even if you don't see the ink by eye... but the sensitive IR-LASER of the machine will detect any hint of blue as strong signal).

Dennis :

We've had good luck using "SeaBlock Blocking Buffer" from Thermo Scientific, cat.#37527.

Ron

We use nitrocellulose and block in Rockland Blocking buffer and get high quality images with little to no background.

I had no problem with the system. Well, what Tobias Bethge pointed out might be be correct, we cant generelize one condition for all samples/proteins, thus it is more likely to be trial and error. I suggest you in parallel to use other system, either peptide/protein-based blocking system (BSA or skim milk) or non-peptide system (here, I would add Roche blocking reagent as an another option). However, in my experience, high background is due to the high concentration of secondary antibody, regardles type of membrane you used. I would suggest you to reduce the ratio as well. On my hand, 1:5000 gave an acceptable background. Again, it is based on my series of trials and error :) Good luck.

Your concern might not be a blocking issue. The LICOR protocol calls for the last wash to be performed in only 1xPBS. Because I was experiencing the same issues as you, I changed my final washes to; 1 wash 1xPBS+0.1%Tween, and then 4 consecutive washes with 1xPBS (4 min/wash) to remove excess tween. Additionally, this removes additional background and allows you to perform you initial blocking steps in milk WITH tween. I 99.99% stand by this protocol :)

5% milk or BSA works best. Though I use 1:10000 dilution for secondary antibody.You can try washing them longer with TBST, say 10 mins, 3 times if you are getting background.

The protocol we got years ago when we bought the Odyssey calls for blocking buffer of 0.1% Casein in PBS, primary AB buffer is 0.1% Casein+0.1% Tween 20 in PBS, secondary AB buffer is 0.1% Casein+0.1% Tween 20+ 0.01%SDS in PBS. When we use this we have very little problems. We block overnight, do primaries 1-1:000 (usually) overnight, and secondary 1:10,000 either overnight or four hours. We wash copiously with blocking buffer. (After primary AB washes are 15min, 30min, 15min; after secondary 15 min, 1 hour) This protocol takes days but gives very good results generally. Sometimes we just use the primary AB buffer (0.1% Casein+0.1% Tween 20 in PBS) for everything and still get very good results (when we don't feel like making all the other buffers).

I have been working with LI-COR Odyssey for last 4-5 years now..and what I observe is that Licor Odyssey blocking buffer or Licor casein blocking buffer works best and you see less background. Have been using this for many years now. As a pilot experiment I did work with BSA and Milk but they gave me very high background. The use of the membrane also determines the use of blocking buffer. The best membrane to use when performing western using licor is PVDF-fl or nitrocellulose. I have been using nitrocellulose 0.22 um membrane.

I really like nitrocellulose for LI-COR Westerns. Low background, good sensitivity. Using an inappropriate, high-fluorescence membrane can cause high background. It's easy to make sure your membrane is compatible. Just wet a piece of unused membrane and scan it to assess the background fluorescence (this is more informative if you also scan a piece of known "good" membrane for comparison).

Some representatives from LI-COR gave a seminar here a few weeks ago. Different brands of nitrocellulose and PVDF have very different fluorescence. Their advice was very similar to Amy's. BEFORE you transfer to the membrane, try a piece in the Odyssey. Also, you can get samples of LI-COR's Blocking reagent here: http://www.licor.com/bio/products/reagents/antibody_request.jsp

Stacy is right. Different brands of membrane have VERY different background levels. It's easy to test the membrane, and will save you a lot of trouble. I really like the LI-COR blocker, but there is no single blocker that works for all primary antibodies. Optimization is the key, if you want really beautiful Westerns.

You need to understand where your background is coming from... whether it is purely a membrane issue, insufficient blocking (too low blocking reagent, which means your antibody starts to bind the membrane) OR your antibody is recognizing a sequence in your blocking reagent. Simply put, you need to try some things out. Load one sample in 10 lanes en cut the blot in 10 pieces and treat them differently (blocking reagents, % block, buffer.......-/+ 1st/2nd antibodies).

Thanks to everyone. I tested various suggestions (PVDF vs. Nitrocellulose; different amount of SDS; different dilutions of secondary ab). In my case Nitrocellulose blocked with 5% milk in PBS-T and sec ab at 1:15,000 in 0.01% SDS + PBS-T was best. Thanks again!

LICOR recommended omitting Tween 20 from the 0.5% casein, PBS that we use.

They say that SDS is not needed with secondaries that we use 800CW and 680RD.

ANOTHER question is how can one get 100% transfer from the gel? We are trying to quantitate extracts and cannot see how to relate the results back to the extract.

@M.H. - The transfer efficiency depends on the MW of the protein of interest. (For example, HighMW take longer to transfer than smaller LowMW. However, because you have the LICOR :), a great, easy standardization assay you could perform is to load

Hi Amy, what nitrocellulose do you use? Do you like to tell the Cat#? Thanks.

We use a selection of three blocking buffers with PVDF-FL in our lab: (1) Li-COR blocking buffer (best), (2) 0.5% filtered casein (OK), and (3) through the help of a fantastic local rep, we managed to find out how to make a block solution that gave almost identical results to the original Li-COR one using fish skin gelatin.. instructions can be found by searching "universal blocking buffer".. such as these ones:

http://www.cytographica.com/lab/solutions/teleostein.htm

http://www.ihcworld.com/_protocols/blocking_solutions/blocking_solutions.htm

We haven't used BSA or Skim milk as we were told it may cause high background. But casein and our universal block buffer works if budget is a factor. Also, Thermo sell an equivalent buffer, but it was similar costs to Li-COR.

Millipore FL blocking buffer is also working nicely.

We used to use Licor blocking buffer which seems very expensive. Now we have been using Aquablock blocking buffer (PP82) from East Coast Bio. Which is way more cheaper and works perfect.

PVDF-FL membrane is a good choice. Blocking with Licor BB or Thermo BB are good but expensive. I appreciate the universal blocking buffer and aquablock suggestions above. Use PBS or TBS with 0.05% Tween20 for washing and dont forget to use PBS or TBS without Tween20 for the last wash. Tween20 has a slight IR fluorescence. The use of a vacuum-assisted washing device e.g. SnapID (Millipore) greatly speeds up your Western blot procedure and improves signal to noise ratio. Make sure that all your buffers are not growing bacteria or mold or have denaturing proteins that precipitate on your blots and cause background.

In you experience with Li-COR, what's the level of "bleed through" you have b/w two color channels? How does that compare to your expectation? on a separate note is the overall accuracy of quantitation higher with Li-COR/FL than blot-strip-blot with CL? Many thanks for your input!

If you block the membrane correctly, the bleed through using the licor scanner is quite low. We routinely used dual colored westerns, but you must block the membrane appropriately (i.e. without tween or detergent in the blocking solution). The quantification we have obtained with the scanner is also fantastic.

Nitrocellulose has a much lower background (esp 700 channel) than even the very lowest background PVDF we have tested. Nitrocellulose lots can vary in background (all low compared to PVDF) and rather than lot qualify these for ourselves we found that simply purchase the LICOR nitrocellulose, who do this themselves, the most cost effective route. As for blocking buffer, the LICOR product is great but is indeed expensive and we are looking for something more cost effective. We will likely give Aquablock a try; thanks Amritlal for the recommendation. 

I am using Rockland blocking buffer with Immobilion-FL (Millipore) and I can highly recommend it!

 We use LICOR Blocker PBS. Some time ago  I bought from Sigma "Gelatin from cold water fish" , but have not yet set up to use it.  I can't remember where I found out about using it.

About PDVF membranes, we bought  what we thought was the usual cut size membranes from LICOR.   What we got was a 3.75 meter roll!  Worst still, it is labeled "for best fluorescence results use by June 15!  My $400 error!  Of course I will still use it after that date!

Personally, I like to use nitrocellulose with the LI-COR system. The background fluorescence is much lower than PVDF, and I find that nitro gives me better detection sensitivity for low-abundance targets.

A tip to improve sensitivity: After transfer, let your membrane air-dry overnight rather than putting it right into blocker. Thorough drying makes the proteins bind more irreversibly to the membrane, so blotted proteins are retained better during the immunodetection process. It really works!

For brain tissue 5% non-fat milk in TBS works like magic

Coming from a classical ECL background loved the PVDF (more robust, lower background)...but NOT with the Odyssey.   Agree with Amy that PVDF should not be used with the Odyssey, especially in the 680 channel where background is just plain impractical  Stick to nitro and optimize your blocking (heavily Ab-dependent; 5% milk in PBS in our jack of all trades), and should work like a charm.

I have bing using Odyssey for about 10 years now.  We use both PVDF and nitrocellulose membranes with excellent results.  The PVDF membranes from Licor are tested for low background or you can by them from Milliport and ask them for membranes compatible with Odyssey, which is a little cheaper.  Some times you need to test the membrane lot by yourself.  We use the blocking agent from Licor diluted by half and it works great.  We use extremely low protein amount to perform western blot. PVDF with the Licor blocking solution diluted work excellent.  The WB will never be better than the antibodies you use.  Be sure to centrifuge your primary and secondary antibodies is you are working at very high sensitivity (low protein or low level of detection).  

Be a little careful with milk, if you're using biotin-streptavidin or phospho-specific antibodies. Milk contains endogenous biotin and phospho-proteins that may increase membrane background.

Yes, every lot of PVDF membrane from LI-COR is QC-tested for low background fluorescence.

I always had excellent results with the Li-Cor reagent.

We use LI-COR's for PBS. with no problem that we are aware of!

Some time ago LI-COR offered a trial kit that included  100 mL batches of their PBS and TBS versions and casein.

We used to use casein, but in 2013 switched. We also bought Sigma's Gelatin from cold water fish, 40-50% inH20, but that was a nuisance to use since it is a very solid gel stored at 4 deg and we never tested .it.  I am ready  to donate the 250 mL. Send address!

I use BSA. Make sure your blot membrane is compatible with LICOR specification.

I suggest referring to my work:

Article Optimization of blocking conditions for fluorescent Western blot

One often over-looked improvement (yet recommenced on the LiCor site) is not having Tween-20 present. Don't have it in your blocking solution and at the end of your blotting process, just before you scan the membrane, rinse the membrane several times in PBS/TBS to remove the detergent. For low abundance proteins especially it can make a noticeable difference in reliable and quantifiable detection.