I have created knockout cell lines using CRISPR/Cas9, and I am attempting to verify that there is no protein expressed via western blot. I Have had no problem detecting this protein in a control cell line with high expression of the protein, but in my cell line of interest it seems that the expression is much lower and more difficult to detect (even the wild-type) without introducing background bands in the western blot.
I Have my reasons for wanting to use this cell line, so I have been searching for a method to detect low-abundance proteins, and someone recommended I use immunoprecipitation to enrich for the protein. I began researching protocols, and now I am wondering if IP followed by western blot would give me a quantitative view at how much protein is expressed in these cells. My concern is that the amount of protein picked up in the IP might be more proportional to the amount of antibody used than how much protein is actually expressed.
I have never performed IP for the purpose of quantifying expression before, so any insights you may have on this issue would be helpful. Thank you.
Of course you can use IP followed by western blot for quantitative analysis. Especially when you habe a control cell line that still expresses your protein of interest and you only want to detect if it is still there in the knockout line. Be sure to use excess amounts of antibody and excess amounts of beads in your IP. You might want to test different amounts of antibody to begin with.
I used IP to detect the amyloid-beta protein in mouse brain which is also of low abundance.
https://www.researchgate.net/publication/285215509_Endothelial_LRP1_transports_amyloid-b1-42_across_the_blood-brain_barrier
If I can help you with any IP protocol, let me know!
Article Endothelial LRP1 transports amyloid-β1–42 across the blood-b...
You can also check (1) some amount of your whole cell lysate prior to IP, (2) some amount of your supernatant during IP for your protein of interest. These will help you find out any loss (if) of your protein. At the same time I believe the whole cell lysate will give you the quantitative protein analysis in comparison to the IP.
Western blot is at best semi-quantitative. you'll be able to say whether there's more or less protein between cell lines, but to interpret bands on a blot by densitometry as quantitative expression values is inaccurate, it is even more inaccurate when you're doing an IP beforehand. Best thing to do is use western blot to see the trend in protein expression and do qPCR for your gene to get quantitative expression values. You can do your IP to see the trends in protein expression for that low expression cell line, you will have to optimize the IP by measuring its efficiency (by loading input, supernatant and IP), but you should absolutely back up your WB data with qPCR if you want truly quantitative gene expression values.
For quantitative results, equal amount of protein from your lysate should be taken and run with IP sample to confirm that equal amount of protein has been taken in the starting. However, after IP followed by western blot should be probed with loading control and/or immunoglobulin chain to show further that equal amount of protein has been used for IP and/or equal amount of bead has been added in all the samples.
Hi Brian, I think your concerns about IP are correct, seldom an IP will capture 100% of the protein in the lysate and of course the amount of Ab used, the affinity of the Ab to your antigen, the kinetics of Ab binding to the antigen and the relative amount of the antigen and the Ab will affect the quantity of IPed protein. An IP efficiency can range from 20 to 90%. I would not use IP to quantify protein in different samples, although it is fair to use IP to quantify co-IPed proteins as a (internal) ratio between the protein you IP and the protein that is co-IPed.
However in your case IP is necessary to concentrate the protein of interest in order to obtain a better, cleaner signal by WB, and the concentration effect of the IP will likely reflect the amount of protein expressed in cells. Ideally you should have no protein in KO cells (I didn't understand if you subcloned your cells or you might have a mixed population: in the latter case be aware that if your mutation affects in any way the growth, the replication or the survival of the cells you might slowly lose ko cells while wt one take over, so the analysis at one point might not reflect the situation after several replication rounds). But even if you happen to have some, it should be substantially less than in control cells, and this should be reflected by the IP. You only have to control properly for the input material, so besides quantifying your extracts, you will load an input control (20-30 ug of your lysate prior to IP) and since your protein will likely give an unclear, weak signal, you should use some other protein to evaluate the concentration of your input and/or stain the gel (also after transfer, or you can stain the membrane after you are done with the Abs) with Coomassie stain.
In any case I support a qPCR analysis of the mRNA if you can design specific primers (for SyberGreen) or probes (for hydrolysis approach) for your mutation and for the wt, as suggested above.
Good luck!
i think if you just want to concentrate you target protein to detect KO efficiency, it won't be necessary to perform IP, as Pietro stated, IP can bring many variations and distract your quantification.
i think a better way is to seperate cell fraction enriched in intrested protein.
plus, if the antibody is not sensitive enough to detect your protein in WB, it may also fail to IP your protein in KO cells or even WT cells
Hi Brian,
I think you can. Just keep in mind that every is proportional and your are ding a semi-quantification (instead of than absolute quantif). So everything being done in the same conditions (same protocol and same concentration of Ab for all experimental conditions you are testing and comparing), the results should then be comparable with one and other.
May be you can improve the level of detection of your protein by suing a more sensitive ECL substrate (for' example Western ultra lightning Ultra' from PerkinElmer, or Femto ECl from Pierce) do a good job with a low increase (especially the Perkin Elmer)of the background.
Cheers
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