I have carried out some nanoparticle treatments on a set of cells, which I plan to use for western blot.
Of 7 treatments, one set of cells has turned red, while another dark purple.
I think this will be problematic for a BCA assay (and bradford), and can't run absorbance since it is a mixed cell lysate.
I wondered if anyone has any other suggestions of how to quantify?
Thanks in advance!!
Brittany
A semi-quantitative way to quantify proteins is to run a small amount on SDS-PAGE with a 2-fold dilution series of a known amount of BSA, then to stain the entire gel with Coomassie, Silver Stain, or Ponceau. You can then take an image and determine density (densitometry) in ImageJ or some other software to build a standard curve with regression off of your BSA lanes, then use that to determine your experimental lanes.
The drawback to this is if you have many bands in your lane, then you will only get a semi-quantitative understanding of what is actually in your sample. The benefit is, you can choose a region of interest (where or your target is) or with a particularly strong band (like the actin region around 45-50 kD), and normalize your samples there.
If you are wiling to spend money, then a fluorescent assay may be a good choice, as well: https://www.thermofisher.com/order/catalog/product/R33200
Thanks Jordan! I think in this case I will probably just normalise based on Actin after running the westerns as I know approximately how much protein I yield from a standard T75 prep. Thanks very much for your advice!
One of the best methods to quantify protein is to use Lowry Method where BSA is used as the standard. Lowry method will give you total protein concentration. I guess this will help. Try to find some research paper on it. If you find any difficulty then do message me, I will send detail procedure of it.
It's also possible to count the number of cells using a Coulter counter, and normalize for that
I advice you quantify using QuantityOne, because you can more easily (compared to imageJ) subtract the background using the rolling disk method. This method is actually possible in ImageJ, wherein it is called 'rolling ball'. In addition, it's best to factor-correct between sessions (between membranes) using for example JM Ruijter's Factor Correction software. This way, you will have less variance between membranes.
Hi you can also try to see whether your lysis content is compatible with the Qubit protein assay from Thermo. This assay is fluorimetric so the colouring resulting from your treatment won't affect the test results. =) We are using it for high urea lysis buffers.
https://www.thermofisher.com/order/catalog/product/Q33211
Dear Brittany:
I would recommend u use pierce 660 assay, and do it on nanodrop. It works perfect for me. Just remember to add Ionic Detergent Compatibility Reagent (IDCR, Product No.
22663). Also it is better to use bioruptor to destroy the DNA to avoid your sample sticky to the tips. Hope it helps.
Best,
Bin
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