I should add that I also have a paper where Bailey's Irish Cream was used as a blocking agent, but so far have not been able to get purchasing to approve the PO.

the only con I can think of, is that Odyssey is damn expensive, and not every department wants to boy it. Otherwise it's the awesomest way to visualize westerns!

I use chemiluminescence with camera based system. I used to use film, but camera based system are more sensitive, flexible and time sparing. I do not have experience with Licor western blot detection.

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June 2018 Update after Using for 1 Year:

Licor in comparison to film:

• Pros:

Easier, faster, double bolting, direct quantification.

• Cons:

More expensive start, less sensitive, automatic algorithms that enhance the signal/background ratio may lead to loss of signal from lower expression samples.

LiCOR-IR based system is obviously elegant system and more quantitative. We also use it for EMSA, Cell migration, coomassie stained gel pictures etc. The costaining option in western is really good. Its also nice that if we couple both chem and IR we can get maximum data from a blot without stripping. But problems could be the cost . Additionally if u are a regular western blot user then all the instruments and vessels could have ponceau, coomassie etc etc, which brings a lot of background in Odyssey.

I use both types of detection. Odyssey is excellent if you want to quantify your protein expression, because you can use two secondary antibodies together (one red and the second one green ) and have a reference protein like Actin.

To add the the support of the Odyssey, ECL is limited to immunoblotting pretty much. In contrast, IR imaging can do several other, similar techniques. For instance, low resolution immunostaining of tissue/cells, in-gel westerns, DNA electrophoresis. The Odyssey uses less secondary antibody, no ECL reagents or film (therefore no wasted under/over exposed film), no dark rooms, no developer and fixer solutions. The initial purchase is expensive, but you probably save on operating costs.

We have LiCor in our lab, however, sensitivity is quite low. I would not invest money into this device if I could go back in time.

More quantitative, not because of two-color staining but licor has a wider dynamic range with more linear response. Film can be easily saturated which make it difficulty to quantify. With licor, you can say you did quantitative western. Used both and not going back to chemi. If sensitivity can be a problem, i think western conditions should be re-optimized unless you're dealing with low-abundance proteins from precious samples.

"If sensitivity can be a problem, i think western conditions should be re-optimized unless you're dealing with low-abundance proteins from precious samples." Namjing Chung

I think that most people do westerns on low abundant proteins, so sensitivity is the major issue. We have tried to adjust assay conditions without any sufficient progress. As for wide dynamic range of LiCor, its truth, unfortunately its starts from quite high quantity and for us is mostly useless. Pity.

Oh, may be we should be using triple-distillated water instead of double one :)

that's weird... we actually once had problem of too much sensitivity in Licor...

Sounds stupid, but it was the case, since it detected differences in stripes, although we used a comb, which is normally used for 2D. We loaded sample into that, ran sds-page, transferred and the we cut the membrane into stripes (we needed to test different serums). However we still saw difference between edges and middle of the blot (not by coomasie or ECL) by LiCor...

I am actually having some trouble with the Li-cor myself, and am looking for good protocols for stripping a PVDF membrane that has a Li-cor secondary, so I can then reprobe it and use ECL. Any advice?

You can't use pvdf membranes with the Li-cod scanner. It has to be a nitrocellulose membrane.

Lara, Stripping and reprobing a fluorescent Western blot is a little more difficult than with a chemi Western blot. Please be aware that a number of the commercially available stripping buffers have been developed and optimized for use with chemi Western blots. These types of stripping buffers will not be effective with LI-COR IRDye because they work by just inactivating HRP instead of completely stripping the antibody.

To start, you will want to make sure that you do not allow the membrane to become dry at any point during your detection process. Time, temperature and concentration definitely contribute to the ability to strip a fluorescent blot successfully. I am attaching a link to the optimized PVDF Stripping Buffer protocol below. Feel free to let me know if you have any additional questions.

Updated Link: http://biosupport.licor.com/docs/928-40032_NewBlotPVDFBuff_988-11933_1364922652.pdf

Dayan, you can use both PVDF and nitrocellulose membranes on the LI-COR Odyssey. If using Nitrocellulose membranes any brand should work just fine. However, if you prefer to use PVDF membranes, make sure to use one that is specific for fluorescence. There is a lot of variation in the production of PVDF from one vendor to another and this can appear as background on your membrane. There have been a number of them tested out and I have attached the results below.

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

Hi Meredith,

I have tried clicking on the link for the optimized stripping buffer protocol. Unfortunately, the link seems to have expired. Is it possible for you to upload a fresh link or is it okay for u to provide the recipe of stripping buffer for fluorescent western blotting?

Thank you very much for your help!

Regards,

Kitson

Hi Kitson, thank you for letting me know about the problem with the link. I have gone ahead and attached an updated link to the information on stripping and reprobing of fluorescent Western blots

http://www.licor.com/bio/products/reagents/newblot_stripping_buffer/index.html

Don't hesitate to contact if you have any questions.

Thank you very much, Meredith for the updated link! Is the content in the updated link similar to the previous one?

For the time being, I do not have NewBlot stripping buffer with me yet. It might take awhile before I can get hold of the item.

If I want to strip and reprobe my recent blots, could you please recommend suitable stripping buffer recipe for nearIR fluorescent western blotting? For your information, I am actually detecting my proteins of interest using either IRDye 680RD Goat Anti-Rabbit or Goat Anti-Mouse secondary antibodies.

I am not sure whether I should use mild or harsh stripping buffer. Appreciate if you can advise. Many thanks!

I used LiCOR system for the first time in 2007, and have not gone back! Awesome technology; no complaints at all. You can strip NC membranes with 0.2N NaOH for 5-10min, followed by 3x10min water washes and re-blocking; it works pretty well for me. PVDF somehow did not do so well, with too many scratch marks and background, and I did not use it after 2-3 times at the begining. You can even use (scan) the peptide arrays (like ActiveMotif's histone PTM peptide array) with dual antibodies simultaneously; I have done it. Two thumbs up from me.

The link that I previously attached actually provides more information regarding the NewBlot Stripping Buffer as I wasn't sure if you were using PVDF or Nitrocellulose membranes. It goes into more detail regarding optimization, such as factors that can affect the efficacy of your stripping and some frequently asked questions for both the PVDF and Nitrocellulose Stripping Buffers. I am providing the direct links to both PVDF and Nitocellulose Stripping Buffer informaiton as well as the optimization protocols below

PVDF: http://www.licor.com/bio/products/reagents/newblot_stripping_buffer/PVDF_factors.html

PVDF Optimization Guide: http://biosupport.licor.com/docs/928-40032_NewBlotPVDFBuff_988-11933_1364922652.pdf

Nitrocellulose: http://www.licor.com/bio/products/reagents/newblot_stripping_buffer/nitro_factors.html

Nitrocelluose Optimization Guide: http://biosupport.licor.com/docs/928-40030_NewBlotNitroBuff_988-11934_1368823939.pdf

I am not sure if you are familiar with stripping and reprobing using fluorescence, but your conditions for fluorescence will need to be more harsh than with HRP.

Hi Meredith,

The third link does not seem to work (http://www.licor.com/bio/products/reagents/newblot_stripping_buffer/newblotNitroDescription.jsp) and the fourth link pull up the Nitrocellulose stripping doc which was useful info. I specifically found your PVDF optimization guide really useful (made sense of my ongoing membrane woes), could you please post the nitrocellulose optimization link/PDF link?

Thanks,

Praveen

Thanks for the notice. I have updated the links above. You mentioned that you have ongoing membrane woes, is this in regards to stripping and reprobing? There are 3 major factors that can affect the stripping efficacy, the concentration of the stripping buffer, the temperature at which you strip, and the amount of time. If you would like to email me your images, I would be happy to take a look and see what I can do to help out.

LI-COR, the C-DiGit Chemiluminescent Western Blot Scanner may be better if you strip membrane often and don't like to use film. Just 5000 $.

http://www.licor.com/bio/products/imaging_systems/cdigit/

we bought Li-cor C-digit scanner but its crap. Its lower threshold is very high.

often the bands detected on X-ray in a 2 min exposer as just missing in it.

we called the technical person fro LI-cor ( some Dr Michal) but it was of no use. he suggested to change the substrate after admitting that signal is below the lower threshold. So even < 5 min exposer on X-RAY is more sensitive that li-cor c digit scanner for low signals

LiCor works very well. Much lower reagent cost/blot than film and the images are collected for quantification at the same time.

I agree with Kunal about the sensitivity. I do not like the Li-Cor mainly due to background/noise signal. Though, company recommends their blocking buffer which I have not tried yet as I used to get a very good signal with Bio-Rad imaging system with normal western blots. May be Li-Cor is good for some antibodies but not for wide range of antibodies.

We have not had any problems with sensitivity or background. Milk blocking is all we use. Make sure not to add tween to the blocking media. I have not had any experience with the C digit scanner, since the idea was to get away from chemiluminescence. Good blocking and efficient washing usually solves many problems with blots.

What percentage of Milk do you use for the blocking buffer Neil?

5% milk, just standard dried milk from the grocery store.

I should add that I also have a paper where Bailey's Irish Cream was used as a blocking agent, but so far have not been able to get purchasing to approve the PO.

we found rockland blocking buffer for odyssey westerns worked very well- low background.   We used it 1:1 with antibody dilution buffer for antibody incubation.  A water wash at the end following final washes also reduced background.  

 Hi could you suggest a suitable way to striping WB membrane for LICOR (IR based antibodies). I used NEWBLOT IR STRIPING BUFFER FROM LICOR but its not much effective. Is it due to membrane dried up during the process. 

Thanks. 

Shivendra, if you have yet to find a good stripping buffer, try out GM Bioscience's OneMinute Wesern Blot Stripping Buffer (Cat no GM6001 or GM6002). We apply it for 1 minute at 37C, wash 2x with PBST and then block again. 

It doesn't always remove all of the antibodies -- I don't think any stripping buffer will -- but it should significantly reduce the signal. You can also try a longer incubation time if you have a very tightly-binding antibody that you really need removed.

Thanks William,

Stripping is good with LiCOR stripping buffer. It takes 1 h for good stripping and removes all the antibody from the membrane. The problem I was facing was due to drying of the membrane while scanning. 

Actually we dry our blots before scanning.  This gets rid of air bubbles, and improves signal:noise

Drying is great for more consistent imaging. Stripping with any buffer using any system (including Li-cor) is highly variable. You sometimes get relatively good antibody removal, lots of protein removal, or just uneven stripping across the membrane. For a qualitative analysis I guess it is okay. For quantitative analysis for an experiment, not so good.

Interesting, I like to wet the membrane and use a roller to remove air bubbles. In this way to make sure the membrane to touch the glass.

Okay so pros and cons to wet and dry. Wet I think you get a sharper image, but it is hard to get rid of the bubbles and small amounts of buffer can alter your image. Dry is very consistent and if you cover the membrane with the big plastic pad you get from Li-cor you get a good image. However, static charge on the plastic pad can alter the quality of your image (static charge does not interfere with wet images). 

The Odyssey is great. Being able to perform two-color imaging on a blot is a huge bonus for me, especially since I need to probe for about 6 or so antibodies. The sensitivity is about as good as or better than film and the single exposure is nice, so I can get multiple images off one run. The analysis program is actually pretty good about recognizing bands!

You will have to go through a bit of optimization, since the buffer you use may or may not interact so well with your secondaries. I use casein to kill off background, but my colleagues all use skim in TBST.

If you are getting background, I'd toss 70% EtOH on the plate and wipe it away with a KimWipe. The machine's 700 channel is especially nitpicky about debris and blue dye. Roll your membrane to remove the excess liquid, blot it away and you're set. Use the silicone pad to prevent it from drying out. Apparently, LiCor says you shouldn't strip a dry membrane.

Their stripping buffer is powerful, but only with ultrapure water. Regular diH2O seems to weaken it by about half in my experience (swapped to ultrapure water with the NitroStripper solution and wound up ripping all of my protein away)

Now, all this praise said, I did stick up for the Odyssey. The CLx model, specifically. I did not stick up for the C-Digit. That thing is less sensitive than film and just kind of meh in general. It's fine if you're checking GAPDH or some incredibly strong antibody, but for the itty bitty expression levels, it's pretty underwhelming. Which is a shame, because I'd have liked to stick to HRP antibodies, since they're cheaper than the IR ones.

This is an old thread but apparrently a catch-all for comments about the LI-COR scanners. I have been using an old Odyssey Classic for a couple years now. One comment. For me the 800nm channel is superior in low background than 700nm. Milk is one of my least favorite blocking agents for background. I use whole serum of my secondaries for best signal to noise. BSA is okay but not as good as serum. PVP40 tests were horrible for me. Secondaries congugated with DyLight 685 and 800 work fine--especially the 800-4XPEG--much brighter. The machine has some bleed through at higher photomultiplier gains, so I try to avoid going above 7 (on a scale of 10). LI-COR now has a 700nm HRP substrate that works fine when imaged wet in TBS. I always image wet, as the 800nm flurophores don't image well dry. However, the DyLight 685 is brighter dry than when wet.. Using a silcone mat and excess buffer on the glass keeps the blot wet and removes bubbles when imaging. Scanning is slow but absolutely no lens abberation as can be with some camera based systems. Bio-Rad and others are finally coming out with NIR capture systems. That should help get LI-COR some competition on it's extravagant pricing.