I have reused conventional transfer buffer (with 10% MeOH, 0.01% SDS) three times with no problem at all--I keep the buffer after use at 4C. I have reused TAE/TBE many times, and I do just as Guido says above! In fact, I have reused the agarose gels themselves! I would have them over-run at a low voltage, and would load fresh samples again! Of course, those were only to quickly see if the digestion/PCR reactions worked. During my early days, I have cut out the band-containing portion of the agarose gel and have re-melted the clean gels and poured into fresh moulds. That made the gel a bit brittle, but that was good enough for low-profile experiments (and, with no funding those days, that saved me some money). About some Ab dilutions--well, I have some bad experiences. In my earlier days as a researcher I have faced disasters owing to my "conservationist" (or, miserly) attitude, and I learnt from those experiences that it is wiser to use fresh reagents while the stakes of the results are high. One advice to readers: never reuse any reagent if the result is of critical importance (and I know people are not as dumb as me to need such advice!). Do not reuse running buffers if you are planning Southern or Western experiments, they might give a higher background. Also, NEVER reuse TAE/TBE if you plan to isolate restriction fragments for cloning/subcloning.

Hi Jan, which buffers or chemicals do you mean? Cheers, Nadine

Most commonly reused around me are electrophoresis/transfer buffers, antibodies.

TAE or TE-buffer f.g.?

Hi, Jan, yes, you can re-use buffers for phoresis, transfer buffers, even antidies. Of course, you can not re-used buffer aftre washing of membrane. To make sure that buffer is work, one can add 10% of the new buffer. .

Hi Jan,

TBE buffer for DNA gelelectrophoresis i reuse for months (add sometimes some water, to equal the evaporation).

antibodies for westerns can be reused sometimes, dependent on the antibody. And also the reuse cycles are dependent on the ab

transfer-buffer for western we reuse one to two times, more reuse cycles decrease transfer efficiency dramatically.

best

GAD

We used minigels which ran for less than 2 hours. So, I have reused SDS-PAGE Running buffer twice, and it worked fine. TAE and TBE buffers also worked well with reuse. I did not try to reuse Transfer Buffer for Proteins.

I have reused conventional transfer buffer (with 10% MeOH, 0.01% SDS) three times with no problem at all--I keep the buffer after use at 4C. I have reused TAE/TBE many times, and I do just as Guido says above! In fact, I have reused the agarose gels themselves! I would have them over-run at a low voltage, and would load fresh samples again! Of course, those were only to quickly see if the digestion/PCR reactions worked. During my early days, I have cut out the band-containing portion of the agarose gel and have re-melted the clean gels and poured into fresh moulds. That made the gel a bit brittle, but that was good enough for low-profile experiments (and, with no funding those days, that saved me some money). About some Ab dilutions--well, I have some bad experiences. In my earlier days as a researcher I have faced disasters owing to my "conservationist" (or, miserly) attitude, and I learnt from those experiences that it is wiser to use fresh reagents while the stakes of the results are high. One advice to readers: never reuse any reagent if the result is of critical importance (and I know people are not as dumb as me to need such advice!). Do not reuse running buffers if you are planning Southern or Western experiments, they might give a higher background. Also, NEVER reuse TAE/TBE if you plan to isolate restriction fragments for cloning/subcloning.

You can reuse TAE/TBE running buffers multiple times .. May be for atleast 3-5 runs (if they are not after a long gap). You can also use SDS-PAGE running buffer atleast 3 times.. It works fine.. The problem is sometimes it gets quite blue if you let the dye run out of the gel. Then using it just once more is enough. You could even re use transfer buffer.. if you are doing your transfer not after a long gap.. Like you could reuse the buffer from previous day without hestiation. But re using more than once might not be very good.

Anil makes a good point on reusing antibodies. It is not a good idea because a diluted protein (antibody in this case) is usually unstable even at 4 deg C. Its good to do a dot blot to find the best protein sample-anitbody ratio before doing a Western blot. For dot blot, you spot protein sample on NC or Nylon, dry it, and then do all the steps in a western. You can make different strips and vary concentration of protein sample or antibody each time. You can even try used antibody, and see if it gives good dots and low background. This is a good, economical way to test if your Western will work...avoids the frustration!

Yes I agree with all the people.

Well I reuse TAE buffer 2-3 times. TBE too 3 times or so.

But never reused the electrode buffer in western blotting.

Can reuse transfer buffer for about month keeping at 4degree, until the buffer is clear.

Blocking agent like 5% BSA, you can reuse multiple times until it loses its turbidity and can add preservative like sodium azide to it and store at 4degree.

Agrose gels can me reused for at least 2-3 times.

Antibodies for western blotting like primary can be reused for 3 times or more. Secondary can also be used 2 times. After developing the blot, if you don't get good intensity of band. Than you can redevelop the blot using fresh antibodies.

Thank you all for the detailed answers. This is a really discovery for me something can be reused. Since usually nothing is working in my lab I have been using only fresh buffers until now;)

Here is a piece of caution:

Never use a pre-used reagent (buffer etc.) while working on something for the first time.

For example, if you just procured an antibody for protein XYZ and you have never used the antibody before, then DO NOT use any pre-used gel/buffer either for running or transfer. Things might look straightforward, but in case you do not see the expected result with the new reagent, you will be in greater trouble figuring out what went wrong.

By the way, Jan, could you please elaborate the "nothing" that is not working? We might be able to say a word or two for the precise good! Best wishes, though!

Personal experience is to re-use some buffers when you are the only one use them. I have read an article mentioned the transfer buffer for western blot can be reuse up to 7 times and the paper showed a consistent result. It is enviromnetally friendly and I started to do it and work well. However, all my fellows (5 of them) like to share my bottle and I lost count commonly. I reuse running buffer before but now I will not recommend it. Yes, most antibodies can be reused as long as you always use them in cold room with some NaN3 in it so bacterial overgrowth will not be a issue. Problem in reuse antibodies is we do not know how many time they can be resued without affecting the quality of the result (it does save a lot of money). I can not suggest other bufferes due to my limited experience.

Dear Anil, my "nothing" means it takes me months or even years to get publication-grade pictures. It does not mean no techniques are working, some of them I have perfectly set-up. Typical mol-biol problems: when my Western starts working, I want to repeat the tissue distribution of my protein. But the next animals I'm analyzing gives opposite results to the first one exp. This is not possible to publish it, but does not exclude this affected expression is true. Simply I don't know what affects my protein level.

Another story are the antibodies I'm buying. Even with the all the tricks I know, the way to have the informative results is a hell. Two of them seems to be working (but recognizing different isoforms of my protein), one was working once and died, next two are giving artifact bands only, additional two bind to all the proteins, but not mine... I have ordered the next 3 monoclonal antibodies...

My protein is extremely poisonous to bacteria. It is even hard to grow them without induction. When sometimes you are successful with growing them usually no expression or a lot of the protein in the inclusion bodies is found... Once I had it soluble was not binding to the nickel, since the tag could be hidden... Every time I have one step successful, it is not possible to repeat it mainly due to the moon phase;)

Consider the possibility that the antibody does not recognize the protein of the animal you used for your experiments. Sometimes the manufacturere claim the antibody will work but does not. Sometimes the protein nhas different posttranslational modifications that will give you different molecular weight band. Blast the protein against other animals to see whether it has the epitope foir the antibody you used. I have experinece that the protein was said to be 90-110kd but i keep getting 60 kd band. Turn out the protein I am looking at was not glycosylated as I expected during fetal life. I took me years to figure it out. I do not provide you an answer but simply agree with your frustration.

hi,

-ponceau red, coomassie blue are good exemple of basic reagent that are reusable..

some also reuses the ECL/chemiluninescnce solution (either home made or commercial)...

- gst or his beads can also be reuses if properly regenerated...albeit the manufacturer does not recommend to do so...of course this increases their sale:-)

for sure...nowwadays, the kit generation is well established and only old researcher...still know how to do basic exp ( a hand made mini prep, preparation of basic reagents???

no matter the quality of the training...I know lab , rich, extremely well funded labs..who purchase/subcontract every lab reagents ..amasing the reagent you find in catalogs...

For agarose electrophoresis and prtein electrophoresis the buffer is reused but for molecular reactions the buffers are seldom reused

Transfer buffer (containing methanol) can be reused multiple times.

Here is the publication that proves it: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685604/

From the principles of electrophoresis, the concept of reusing a electrophoresis buffer is wrong.  The more important aspect is defining what one means by "working".

1.  Protein/DNA electrophoresis buffers are specifically formulated to contain particular ratio of cations and anions. Firstly, during electrophoresis these ions are consumed.  They are discharged at the electrodes, and thus their concentrations drop with electrophoresis.  It is IRREVERSIBLE.  An then the voltage and current sustained by the buffer changes.......  next time the run will be very slow.

2.  The cations/anion ratio and even the type (phosphate, Na/CL etc.) create fronts that determine the rate of migration of a protein.  As the ion concentration changes, these fronts change and the protein migration changes - in fact the width of the front or its sharpness are critical to the tightness of a protein band and hence band resolution.

3.  As this ion concentration changes, also a good buffer is made to prevent a drastic pH change across the run.

4. So if you really want to use the word "working" one must show that the plot of migration distance vs log (MW) is a straight line, AND THE CALCULTED MW for the protein from this plot remains unchanged.  If a protein changes from 50,000 to 51,000 that is 1,000 worth of amino acids or ~ 10 aa.  If you publish a sequence that has a difference of 1 Kda then you have some explanation to do......so keeping that in mind one needs to apply the same sense of quality control.

5.  Now consider that your protein has a few more basic or acidic residues than usual.  How will this change from run to run??

6.  In the case of Western blot transfer buffer, as the ion concentration changes the amount of protein transferred FOR A GIVEN AMOUNT OF TIME, will change, because the current changes.

7.  You can adjust the current and voltage, but then the pH of your buffer will change from run to run as your buffer ions are depleted.  Thus, the residual charge on the protein changes, and the binding to the membrane is affected.  If you protein binds less strongly to the membrane, you could easily affect the amount bound.....since both rate of migration (point 6) AND ionic nature changes.

8. Now if your antibody is good the band signal will seem to be OK.  You just expose the film a little longer, and Voila ! you have good bands......But what if your protein has a slightly different charge than normal, or binds to the membrane less strongly.  Now what happens?  You then wonder why your band signal is going down? 

 9.  Kateryna's paper shows the result for a very well behaved protein - p53 - in Western blotting.  I can see many reasons why that works.  But would you want to gamble on a lesser known protein all the time????

10.  So in my view, if you want to GAURANTEE a run will work for ANY protein you try, the make fresh buffers.  Its a small amount of work, for saving a large amount of headaches !! :-)

Dear Prem,

In my lab re-use is strictly forbidden. I have asked this question long time ago to collect all the concrete arguments against it like yours. Was surprised most of the answers were saying re-use can be done! My concern is how many articles are showing the gambling you nicely described….

Regards - Jan

Hi Jan,

Thanks for the reply!

When one loses sight of the "first principles" of any method, you do not understand what you are changing and why.  A 'protocol" is set of fixed instructions that are linked together so that ALL steps work in an explainable fashion.  If you change one part, you better be sure that the other linked parts also work.  Just my 2 cents.......  :-)