I am concerned that using a Lowry assay may not capture all protein content in a ligament sample. Any suggestions for a method? I will be using total protein to standardize a Collagen 3 ELISA.
Hi Amjad, if the protein is soluble then standard protein assays (Lowry, BCA, OD 280 nm, etc) will be fine.
If the protein is insoluble, there will be problems both for protein determination and ELISA.
Ligament sample seems to be difficult as skin sample, which aggregates or becomes to gel at low temperature (please see file; HPLC-Surf-SEC protein determination). Fresh ligament samples should be diluted as described in this article. However, CV (Coefficients of Variation) of data of skin seems to be relatively large even in this reliable HPLC-protein assay method.
Therefore, we have developed the quantitative PDMD (Protein-Direct-Microsequencing-Deciphering) method (please see file; HepG2 Fucoidan).
Component protein of ligament are searched among my protein database; i.e.,
Fetal hepatocyte Hc has Collagen alpha-5 (VI) chain at 3.9 μg/mg cell protein.
Hepatoma HepG2 (cultured without fucoidan) has no such proteins.
Healed hepatocyte HepG2 (cultured with fucoidan) has Collagen alpha-1 (XVIII) chain at 9.5, and Collagen alpha-3 (IX) chain at 0.61 μg/mg cell protein, respectively.
Normal liver tissue (with pseudo-liver cancer) has Collagen alpha 1 (VIII) chain/Endothelial collagen at 5.9, and Collagen alpha 2 (IV) chain at 16.0 μg/mg tissue protein, respectively.
LC tissue (with leprosy) has Elastin/Tropoelastin at 16.6 μg/mg tissue protein.
HCC tissue (with PBC) has Collagen alpha-1(V) chain at 16.3 μg/mg tissue protein.
LC tissue (named No.6) has Collagen alpha 5(IV) chain at 12.5, and Collagen alpha 1(II) chain/Alpha-1 type II collagen at 11.1 μg/mg tissue protein, respectively.
HCC tissue (No. 6) has Collagen alpha-1(IX) chain at 4.4, and Collagen alpha-1(XV) chain/Restin at 1.4 μg/mg tissue protein, respectively.
Serum of 4mo boy (with biotin deficiency and alopecia) has Collagen alpha-6(VI) chain at 10.8 μg/mg serum protein.
Serum of healthy 1y4mo girl has Collagen alpha-1(XVII) chain/120 kDa Linear Ig A disease antigen at 12.8 μg/mg serum protein.
Serum of Gait disorder female (32y) has Collagen alpha-6(VI) chain at 5.5 μg/mg serum protein.
Serum of sister (22y) of the above patient has Collagen alpha-1 (XVIII) chain/Endostatin at 9.1 μg/mg serum protein.
Interestingly, these proteins are up-regulated by HPV, HHV-3, HHV-5, and HHV-6.
Thus, gene expression is under the control of virus except fetal cells.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA