I'm knocking out a 29 KDa protein from human lung epithelial cell line(A549) aiming to make a clonal KO cell line , my target gene has 3 exons with functional domain in exon 2 so i desgined 4 gRNA constructs targeting ex.2 , i selected 2 of them that are working effeciently , I made the single cell cloning using those 2 gRNAs and screened it by first PCR using primers flanking the gRNA binding sites , I got some cell clones having (247bp) deletion which show smaller PCR product with no any larger WT bands, This deletion was confirmed by sequencing n since there is no WT band in PCR so i consider as homozygos deletion.
I did the western blot for those clones using abcam antibody against the C-terminal of my target protein (They said that is raised against synthetic peptide of my target protein) , But there is strong WB protein signal in all clones ?!!!!!!!!!!!!!!!!!
As a control , i did the same experiment but in another cell line , called HAP-1 cells , which is Human haploid cells so in this cell line , the CRISPR KO should be always homozgous , However it end up that i have cell clones with different deletion size and protein is still detected in all of them also.
Knowing that this gene codes for a cellular enzyme , i widely separate the cell clones from the wt during WB to avoid any possible contamination or leakage between wells and i did it for 3 times with the same results (The protein is still there ) !!!!
Has enyone experienced a problem like this before , It will be very nice if somebody would help me solving this issue.
I had the feeling that it is antibody related problem , but this is the only commercial AB available , how can i confirm my protein KO?
Thanks in advance
Mohamed
Is the band in your knockouts the same size as in a WT control? You may have an internal promoter somewhere in an intron that allows production of the C-terminal region of the protein despite having knocked out the N-terminal portion.
It's also possible that your antibody is cross-reacting with another protein (as you suspect.) Perhaps you can consider assaying the mRNA produced by your gene, either by Northern blot or by RT-PCR, to determine whether your gene is being transcribed. If it's not being transcribed, it's very likely that this is just cross-reactivity of the antibody. Alternately, you can add a C-terminal Flag tag to your protein and blot with an antibody for the Flag tag. Because any cross-reactive protein is not tagged, your band should be absent in your knockout. A final option, you could extract the band and run mass spec on it to identify the protein that's being bound by your antibody.
Thanks Sarah for your suggestions , I got a commercial KO cell line where my target gene is deleted by CRISPR also , i lysed the cells and did the western blot again using my Ab , and i got a very intense WB signal at same size , which definetly now means that the Ab binds or detect something else , Also Abcam has removed this Ab from their website
I completely agrees with Sarah.
Also you can try out for complete knockout of your target gene by using two gRNA approach rather than single gRNA approach. For this you can design two separate gRNA one each from N terminal and C terminal ends. It will rule out the possibility of any expression from C terminal end. After this if the protein is still present then second approach you can try out to insert stop codon/termination sequence to rule out the possibility of any expression.
Hope this works for you.
Best Wishes
https://www.nature.com/articles/s41592-019-0614-5
This phenomenon is very common as detailed in the paper above.
since your protein is an enzyme try to test the function of your enzyme. sometimes the protein levels using WB will show no effect while the function is being lost
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line