I extracted total proteins from tissue to do western blot. Some of my samples are in low concentration. Since I need to run equal proteins for SDS page, I want to increase the concentration of them. I have done freeze dry and acetone precipitation but I think it is not suitable for the buffer that proteins dissolved it, can anyone suggest other ways?
Try a centricon centrifugal filter with a molecular mass cut off less than that of your protein. You can reduce the volume without loosing any protein. This also will not denature your sample (if you are careful).
Try a centricon centrifugal filter with a molecular mass cut off less than that of your protein. You can reduce the volume without loosing any protein. This also will not denature your sample (if you are careful).
Tnx George .what is the maximum time centrifuge with these tubes? long time is not harmful for filters? i need small and big size protein in my samples
Check the Millipore website for specific info.... (promise I am not a product rep..)
You simply need to choose a molecular weight cut off less than that of the smaller protein. Then spin until the sample volume has been reduced sufficiently. This can take some time, which is ok as long as you do not over do it and spin all of the buffer through. I have gone as far as ~90% reduction in volume (or 10 fold sample concentration) with no problems. This technique also will not concentrate the buffer/salt the way that drying would. Good luck!
I have done once, but i think with long time centrifuge some proteins loose in this way ,also precipitate with acetone does not have good result , what is dialyse? and how i can do it? tnx for comments
Dialysis is not useful in this case since it doesn't concentrate your proteins.
Millipore Centricon 500ul, 5000ul and 15000 ul concentration tubes will help you concentrate the diluted sample. You need to use 3k Molecular weight cut off tubes.
Please go to Millipore website or Thermofisher. All the best.
RKG
Acetone precipitation is the best choice for your similar [protein] problem or objective for SDS-PAGE. Once you get the pellet...make sure it does not dries out otherwise that sticky pellet wll be hard to dissolve in your 1X loading buffer/dye. Then, during the heating up before loading the proteins should finalise to dissolve.
I don't really know what your incompatibility with your buffer may be, but if that were the case centricon (+ acetone precipitation combined if required) is doable.
Good Luck!
Trichloroacetic acid is the best. In all ultrafiltrations/dialysis steps you will lose up 50% of your protein and thats will happen in an unpredictable manner. Mostly high molecular weight protein stick much better on those membranes. In the precipitation (acetone as well) you will lose mostly very small proteins and peptides.
We have used Millipore centrifugal filter units with a cut off weight smaller than our protein of interest with good results. Also we have used ammonium sulfate to precipatate out the proteins and resuspended in 2% SDS buffer.
After trichloroacetic acid or acetone precipitation, which buffer i can use to dissolve the pellet?since i need to store the protein samples for gradual usage , may i dissolve it in same buffer ?(protein extraction buffer) or directly dissolve in loading buffer?if so , can i store it?Tnx
Another problem is, once we concentrate the protein , buffer will be concentrated as well ,so how can i reduce it?
You can also try lyophilizing the samples in a sample concentrator
There is many methods for this , but the easiest is by using centricon centrifugal filter,
you need to buy this according to your protein size, like if your protein is of 70 kda and you have contamination of 20kda use cytoff filter of 30 kda
you can check this for product detail and information,
http://www.millipore.com/catalogue/module/c3043
order according to your protein size, I think this is good way... i think this will help you.. go through the given below for more detail...
http://kirschner.med.harvard.edu/files/protocols/Millipore_Centricons.pdf
;)
You may want to consider ammonium sulphate or PEG precipitation. Although alcohol is in some Western transfer buffers, alcohols, acetone and chlorophorm are commonly used to crosslink and fix proteins. That may not be good for the final SDS-PAGE sample you are trying to fractionate. Fortunately one can easily compare different precipitation methods.
Thanks for your suggestions , actually i need to measure the concentration of proteins for sds page . how can i measure it after dissolve in loading buffer?
I would recommend measuring protein concentration prior to addition of Laemmli buffer. Detergents and reducing agents may interfere with protein concentration determination. But if your proteins are already in loading buffer simply use the loading buffer combined with whatever buffer your protein is in as a blank to determine background.
Hello there,
one of teh commercially available spin columns will do the job nicely. Acetone precipitation usually works though.
Hello,
Some times the low amount of proteins arises from insufficient cells disruption.
Are you sure that you have disrupted the cells enough?
which method did you use for disruption?
It is possible to concentrate the protein precipitated by contricon or should be completely dissolved ?
Dear all, I found this discussion today, that helped me a lot. I'd like to concentrate a few microgram protein in 100 ul. Centrifugal filters can't help me, because basically this small amount is the lost of that process, so no protein at the end. I will try the powder PEG or Gel Absobent. My collegue suggested me to use SpeedVac to remove the additional water and concentrate the sample. It might take some time, but it is a very confortable method, if you have a SpeedVac inyour lab. Good luck!
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