I have used the same anti GFP antibody several times now for western blotting. I collected after primary incubation and kept at -20. The antibody was in TBS-0.1%Tween-20 and 5% non-fat dry milk.
I attached the blot below. I am wondering if the highest bands are showing any artifact from antibody breakdown?
The problem is that this all should be in true solution and some of the bands look fine.
Please share knowledge and opinions about collecting and re-using the primary antibody for western blotting.
The attached blot is use #6 for the antibody in question (Roche Anti-GFP Anti-Green Fluorescent Protein Stabilized antibody preparation Cat. No. 11 814 460 001)
You may re-use primary antibodies until your signal is dropping too much. Use a suitable positive control to demonstrate your antibody is recognizing what you're looking for and to estimate the signal strength. It doubles as exposure guide for film and cameras.
However, I'd not freeze antibody solutions, as most proteins will denature. Instead, add sodium azide to 0.1% m/v as preservative and store them in the fridge. Is good for years. And make sure all azide is washed away before your blot gets in contact with peroxidase.
Hello Neal Edward Beeman
The biggest risk in reusing primary antibodies is microbial contamination. Antibodies for Western Blotting diluted in solution of 5% skim milk is a big concern when storing the primary antibody dilution for re-use on blots because of microbial growth. To avoid unwanted microbes, you can add sodium azide (0.02%) if you are storing your primary antibody dilution at 4°C for re-use. If you are storing the primary antibody at -20 degree C, you need not add sodium azide. You can reuse the primary antibody solution three to four times as milk will eventually go bad. Another issue is repeated freeze-thaw cycles, which can denature the primary antibody.
As an alternative, diluting in 5% BSA solution would be a good option. BSA functions as a protein stabilizer, allowing you to reuse antibodies longer on storage.
Best.
Hi Neal Edward Beeman .
Your antibody can easily detect GFP in this figure. In most labs, working solutions of antibodies (diluted) are used repeatedly until the signal starts to fade away. I, myself used to use the same antibody (against GFP and many others) at least 6-8 times. As you mentioned, I would not recommend that you freeze your working antibody solution at -20 degrees. This would significantly affect your antibody stability, life, and affect the quality of your blots (will certainly increase the background or reduce band intensity for target protein). Instead, add Sodium azide (at 0.1%), mix well, and store at 4 degrees till further use. But try to use this antibody within 7-10 days, since long-term storage is not advisable. Hope this helps you.
Repeated usage of primary antibodies with success depends on several factors, including
· specificity, binding affinity, stability and concentration of the antibody used;
· and also, the expression level of the protein of interest.
For proteins abundantly expressed, for instance, housekeeping proteins like alpha-tubulin, repeated use could be okay. If your GFP is overexpressed that is fine.
But repeated cycle of thawing and freezing of the antibody solution will definitely compromise the quality.
I would say better to aliquot the antibody solution in small volumes and use one at a time. You can cut the membranes into small sizes based on the molecular weight(kda) of your protein to fit for small volumes of the antibody solution.
It is also highly recommended to run protein marker(ladder) along with your samples so that you would get a good idea whether the observed band is the right protein band or some artefacts due to non-specific binding.
Hi
I have used the same antibody for about as many as 15-20 westerns (like housekeeping GAPDH/Actin) while others antibody may last for about 5-6 times only and often needs spiking with fresh antibody or you may make a whole new antibody. The whole crux of the use and reuse of the antibody is signal detection. Obviously, you would want to make antibodies (in milk or BSA) according to the datasheet of the antibody.
Looking at your western it looks like you are using too much secondary antibody. The dumb-bell-shaped appearance of the band is usual when the too concentrated second antibody is used.
"for storing in 4 degree, will antibody be denatured as well?"
Normally not, unless it's a glycerol stock. Working dilutions with carrier proteins (e.g. BSA, Casein or non-fat milk) are fine, as long as you include a bactericide, e.g. sodium azide to 0.1% m/v
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