I have THP-1 cells differentiated using 20ng/ml PMA and incubated in 12-well plates at 2x10^5. I would like to lyse the cells and extract LL-37 or hCAP18 from my cells to use for an human LL-37 ELISA kit.
I have zero experience with protien extraction so I'm on the deep end with this one. Reviewing literature and consulting with people that have worked on protien extraction has led me in several directions for possible approaches.
I have gotten recommendations to use RIPA lysis buffer, UREA lysis buffer or just cooled PBS for the extraction. I've been recommended to use repeated freeze-thawing or a cell distruptor.
Do I need to use SDS page(gel) to confirm the presence of the protien in solution?
Easy to say I could really use some guidance from anyone that has experience with this particular extraction and ELISA procedure.
Much appreciated.
Hello Prince Dee
Wash the attached THP-1 cells which have been differentiated gently with pre-cooled PBS, and detach the cells using 0.25% trypsin. Centrifuge the cell suspension for 5 min at 1000×g. Discard the medium and wash the cell pellet three times with pre-cooled PBS. You may add 150-250ul of precooled PBS per 1×10^6 cells to keep the cells suspended. Carry out repeated freeze-thaw cycles by freezing the cell suspension in the freezer and then thawing the cell suspension at room temperature or 37℃ until the cells are fully lysed. This method of lysis will cause the cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Centrifuge the resultant lysed cells for 10 min at 1500×g at 2-8℃. Discard the pellet and collect the supernatant to carry out the assay. Add protease inhibitor such as PMSF at 0.1 - 1mM in the final volume during cell lysis to prevent protein degradation.
Please note: Do not use RIPA lysis buffer or UREA lysis buffer since there is a possibility of causing deviation due to the introduced chemical substances.
Do I need to use SDS page(gel) to confirm the presence of the protein in solution?
No. But you need to perform the total protein assay (Bicinchoninic acid (BCA) assay) to determine the protein concentration of your lysate. The volume of each sample can be normalized to deliver the same amount of total protein for the assay.
Good Luck!
I agree with Malcolm Nobre, however you could include a 15 sec sonication step if possible before centrifugation, for better detection of membrane bound proteins.
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How do I extract viable protien (LL-37) for ELISA from attached THP-1 cells?
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To extract viable LL-37 protein from attached THP-1 cells for ELISA (Enzyme-Linked Immunosorbent Assay), you can follow these general steps:
By following these steps, you can effectively extract viable LL-37 protein from attached THP-1 cells for ELISA analysis, allowing for the quantification of LL-37 levels in response to various stimuli or treatments. Adjustments to the protocol may be necessary based on specific experimental requirements and the characteristics of the THP-1 cell line.
It is important to know the location of the protein before structuring an efficient lysis protocol. Subcellular localization is a critical subject to assess before targeted protein extraction. Membrane-bound, cytosolic, extracellular, organelle structural, nucleus, peroxisome, cytoskeleton, etc... Since you indicated a lysis requirement, assuming it is intracellular a physical disruption method with a mild buffer with physiological pH such as PBS or Tris is worth trying.
RIPA-like recipes or other mixtures including SDS may be too aggressive for your protein and any detergent-based lysis buffer may not be necessary. You will be unable to use the protein even if you extract well by employing detergents owing to the detergent-rich environment which may cause denaturing (stated for ionic ones mainly). You should get rid of detergents for the following experiments and this is a sort of trouble for many scholars.
Urea on the other hand is required for improved solubility, other than inclusion bodies or highly hydrophobic proteins such as membrane-bound ones may not ask for chaotropes. Thus expression levels, 3-D/folded nature, solubility (hydrophobicity), and location must be well-documented before selecting lysis application.
Since you are not targeting the whole proteome, Lysozyme treatment and or PBS combined physical disruption (depending on which equipment you have to perform such as; ultrasonication, bead beating, french-press, asprite-dispense cycle through syringe needle, dounce-homogenization, etc..) would be enough. Freeze-thaw cycle process on the other hand a bit risky in terms of making your protein degraded or aggregated therefore remain it as the last choice in my opinion. Do not forget to add protease inhibitors and nucleases (phosphates if this is phosphorylated) to keep the protein intact and to adjust the viscosity of the lysate amenable to the next application
For the question on SDS-PAGE guidance, several proteins with the same MW of your protein can produce identical bands, and this can be assumed as puzzling for protein identification. I would prefer either ELISA, WB, or HR-MS for the validation.
Good luck...
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