Hi everyone!
As I previously shared with you, I've started to work with Caco-2 cells and even asked for your help. I recently followed a protocol to extract proteins, but when I collected the supernatant, it was very mucus-like, and didn't know if it is normal or not, or if I had to do something different from the protocol that I followed.
So far I haven't found anything related to this matter that could help me. Do you happen to know if I have to do something different when it comes to extract protein from Caco-2 cells? Or is the mucus-supernatant normal? Can I do Western Blot with a sample like that?
Could it be because they are a cell line from a carcinoma? Or cause its differentiation into Goblet cells? I have never had this problem with IEC-6 cells, so I'm really lost
Thank you so much for reading!
Can you provide any information on the protocol that you used? Without some additional information it is difficult to make recommendations. It sounds to me though like you are having problems with the DNA content of your cells making your samples gloopy (especially a problem if your lysis procedure ruptures the cell nucleus). This can be more problematic with certain cell types compared to others.
There are several potential ways to deal with DNA causing increased viscosity, depending on the protocol that you are using. Firstly, and probably the simplest option if you are using mechanical or detergent based cell lysis, is to add DNAse to your lysis buffer to degrade the DNA. This should reduce the sample viscosity. Another good way is to do a few very brief (1 second) pulses of sonication on ice at a low to medium power with 5 to 10 seconds between pulses to mechanically fracture the DNA. Passing through a 27 G syringe 10 to 20 times to shear the DNA also works well. Sonication and needle shearing can be problematic though if you have detergents in your lysis buffer as they will cause frothing unless you are very gentle.
If you find the gloopiness appears after heating samples in loading buffer for SDS-PAGE, applying a brief sonication and then spinning the samples down at 10,000 G for 5 minutes so you can pellet any insoluble material and take your sample off the top for loading on the gel is a good way to avoid loading aggregates that will be unable to enter the gel. Although you can skip the sonication and just spin them down, it does help to get things into solution that otherwise may be unable to enter and separate.
Hi Melissa Tirado I've being working and extracting protein content of Caco-2 cells for 4 years, and using that stracts for quantification, WB, arrays... That viscous element that you metioned, is so probably DNA on your sample. That means you are probably using a too "strong"method for cell lysis, unless u want to extract nuclear proteins.
If u just mant to obtain cytoplasmic proteins, using the lysis buffer: Tris 5 mM, NaCl 70 mM, Triton X-100 0.05% (v/v), incubating the cell with it on agitation till it release from the plate, and followed with a mechanical fragmentation (25G needle ), is enough. Finally you centrifugue the sample, discard the pellet and you have the sample ready for any assay you want.
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