Hi everyone,
Has anyone used dialysis tubes for antibody purification? I would like to know if it is possible to reuse these tubes using the same sample and if so, how to perform the washing to avoid contamination.
Dialysis tubing is cheap, antibodies are expensive, and your time is valuable.
Don't bother with reusing such an inexpensive product.
I agree with Katie Burnette. The only exception would be if you were planning to use the same tubing with the same antibody immediately, but as a rule, regular dialysis tubing is not recycled. The situation is different with large ultrafiltration cassettes, which are expensive and can be recycled after extensive washing according to the instructions of the manufacturer and stored in 20% ethanol to prevent microbial growth. With respect to contamination, it is woth noting that dialysis tubing is made of cellulose, a substrate that many bacteria and fungi will happily use as as growth substrate. It is therefore avisable to keep stocks of dialysis tubing sterile (it can be autoclaved) or in the presence of 20 % ethanol.
Dear Yuri
i suggest to you to replace the dialysis with desalting (PD_10 coloums) and if necessary pre-concentrarion of the samples with centrifugal ultrafiltration devide (eg ultraamicon )with 50Kda cutoff membrane which is very fast.
If correctly used and stored you can re-use for the same sample, the PD-10 coloumn an the Ultrafiltration many times. You can just, whash it with milliQ water and store them at 4°C in 20% ethanol.
If you are interested to have more information about desalting and PD-1o gravity coloumn you can see the following 2 presentation avialable on my blog ProteoCool
- buffer exchange methods overwiev
https://www.blogger.com/video.g?token=AD6v5dzeX21U_Er6jBChQIhE5zWtQhNjRjNXRPR7qk7KfWWSQxhf8PbUn4R6KLL0pn_7tU92M0-B3kEUHO9IkTpelmdO79QR53uSg29ZFV3j5D-siHAamTu1y2wOEQ8S4As_wpxzLvA
- rapid buffer exchange by PD-10
https://www.blogger.com/video.g?token=AD6v5dwNlVHz3BclDT9RLY-6KbEEqkS3RnRdHn4BqTsZW4j_prf3KZwHDMTzRYT-B9Yifvmde7RdExL5AArLFtg1Q13fu4-83o3M8bCka1jJ5PfWFK8B53BOpx6YrzvLr2qDiPgT15c
ciao
Manuele
Dr. Manuele Martinelli is it possible to use PD10 mini trap G-10 columns with an exclusion limit (Mr 700) for desalting the peptide extract if the target peptides have MW ranges between 4kda and 6kda. Do you have any experiences based on peptide purification by their weight with these products or can you suggest any alternative?
I am looking for an option to effectively and practically desalting the peptides after the protein removal performing either by precipitation or ultrafiltration.
Thanks in advance
Katie A S Burnette , Pierre Béguin and Manuele Martinelli thank you all for your help in my research. I appreciate and learned alot from your answers. :D
Dear İsmail Emir Akyildiz
you can certainly use it.
However is important that you have an assay to readly detect the peptide i and make the pool only with the fractions those contain it.
For example:
With protein generally i'm using the bradford assay mixin 10ul of eluted sample with 100ul of bradford reagent 1X and i make a pool of the blue fraction. It is feasible only if the protein concentration allow to detect the protein with Bradford Assay. Alternatively we can also use monitoring of A280 (eg with nanodrop).
Ciao
Manuele
Dear Manuele Martinelli,
Thank you so much for your response and,
I apologize to all contributing authors for interrupting the current topic of discussion.
Emir.
Hi,
My suggestion for quick desalting/buffer exchange is to use small prepacked desalting columns, for example BabyBio Dsalt 1 ml where you can use sample volumes 0.02 - 0.3 ml or BabyBio Dsalt 5 ml where the samples volumes can be 0.1 - 1.5 ml. You can use these columns many times and you do the desalting/buffer exchange in a very short time that in many cases is very important especially when working with sensitive proteins/peptides that easily degrades. These column are used either with a simple syringe, a pump on the bench or a chromatography system. this means that you have control over the flow and not using gravity flow. These columns can also be added in series if you need a larger column for larger sample volume. I have attached the instructions so you can read more and you can also check this web site: www.bio-works.com
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