I did protein precipitation with ethanol. now two questions raised :
1. is it OK if i add water to the sample and use it for ELISA and T-cell assay?
2.how I can determine protein concentration after precipitation?
any comment is welcomed!
Ethanol can alter measurement of some cytokines by elisa. Few people studied effect of ethanol, and found that it alters the conformational state of the protein, some even found that protein was degraded, so ethanol prep is not first choice for precipitation. If you make 100 fold dilution of your prep which you have in buffer of your choice against blank, I think you would be able to estimate concentration.
There’s any particular reason you prefer precipitation? If you have your his-tag, go for a affinity chromatography using a nickel column. Using the column will leave you with a protein in solution relative concentrated, use an aliquot to assess
concentration using a comercial kit and/or Optical density.
If using the column, an ELISA should be straightforward just make sure you dilute the sample in the correct buffer. For T Cell assay, just make sure your protein is endotoxin free or all the data could be altered by LPS.
Most proteins will be denatured by ethanol and wont dissolve after precipitation. This is only useful for methods that can accept denatured proteins, such as SDS-PAGE. Thus other approaches are highly recommended and if you can share more information about your experiment i am sure you can get more ideas from RG community.
Mahesh Chandak Thank you Mahesh. what if the protein is with 0.1% SDS?
since I need to solve the protein in 0.1% SDS after precipitation. does it affect the quality of Cytokine evaluatio?
Hi Matindokht,
0.1% is not super high, and I dont think that it will affect. In previous studies an excellent match of sds treated ( 1 as well as 2%) and untreated samples were observed, however this is biochemistry and for each monoclonal antibody test of applicability is the best solution. For your study, I suggest to include control of BSA and any other houskeeping protein of your choice with and without 0.1% SDS as a positive control and measure absorbance in order to satisfy reviewers.
Marcos Javier Ramos-Benitez thank you Marcos. actuall I need this protein( His-Tag Protein) for cell culture. I used Nicole column with a buffer contains Imidazol and 8M urea. then with Ethanol I removed Urea and Imidazol however pellet after precipitation is unsolveable. I am wondering what if I add SDS 0.1% to solve the pellete and go for T cell assay and Elisa?
Mahesh Chandak Hello Mahesh,
thank u for the nice suggestion . I‘ll add control to the experiment.
Markus Sack Thanks Markus, I need to use ethanol method to remove imidazol and 8M urea.
In that case i would avoid SDS at all costs because of direct effects on your cell culture. Also, you need active, i.e. properly refolded protein as you most likely want to stimulate your cell line. Therefore you may rather look into ways to fold your protein on-column, e.g. by changing the buffer. Here are some leads:
Article On-column refolding of an insoluble histidine tag recombinan...
https://www.gelifesciences.co.jp/catalog/pdf/18113437ac.pdf
You can also consult the Qiagen Handbook or similar resources.
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