Detecting the matrix and the machinery that grips it: why a collagen i antibody, and the collagen antibody, collagen type i antibody, collagen 1 antibody and anti collagen antibody spellings sold beside it, has to state whether it sees the triple helix or the denatured chain, what a col1a2 antibody, a col3a1 antibody and a collagen ii antibody separate in tissue, how a fibronectin antibody, an fn1 antibody, a laminin antibody, a periostin antibody, a heparan sulfate antibody and a hyaluronic acid antibody require extraction conditions a cytoplasmic protein never does, where an integrin antibody, a talin antibody, a vinculin antibody, a paxillin antibody, a fak antibody and a cortactin antibody mark the adhesion rather than the matrix, what an icam-1 antibody, a vcam-1 antibody, a pecam-1 antibody and a cd144 antibody report at the endothelial surface, and how an n cadherin antibody, an n-cadherin antibody, an ncam antibody, an ncam1 antibody, an l1cam antibody, an epcam antibody, a cxadr antibody, an occludin antibody, a zo-1 antibody, a claudin-5 antibody, a claudin 18 antibody, a claudin 18.2 antibody, a cx43 antibody and a connexin 43 antibody each mark a junction rather than a cell type

Getting matrix and junction stains to work

1. Decide native or denatured before buying. Fibrillar matrix proteins present very different epitopes assembled and unfolded. A reagent validated on a denatured chain may see nothing in tissue, and one raised against the assembled form may see nothing on a blot.
2. Extract for the compartment. Cross linked matrix and junctional complexes are not solubilised by a standard buffer. Sequential extraction, or a strong denaturing buffer with the loss of native epitopes that implies, is what the blot requires.
3. Choose retrieval per reagent, not per tissue. Junction proteins in fixed tissue frequently need retrieval to be visible at all, and the optimum differs between reagents. Optimise on a tissue known to be positive rather than on the experimental material.
4. Read junction stains as continuity, not intensity. What matters at a junction is usually whether the staining is continuous along the contact, not how bright it is. Scoring intensity where continuity is the biology misses the effect.
5. Confirm the matrix is yours. Cultured cells sit on a coating and in serum containing medium, both of which contribute matrix protein. A no cell control on the same coating shows how much of the signal came with the plate.

Insolubility is the defining property

The biological point of matrix is that it does not dissolve. Every technical difficulty in measuring it follows from that: incomplete extraction, epitopes destroyed by the conditions needed to extract, and quantitation that depends on how hard you tried.

State the extraction method with any matrix result. Two laboratories using different buffers are not measuring the same fraction, and comparing their numbers is not meaningful.

Adhesion is a complex, not a protein

Adhesion structures assemble and disassemble, and their components are present whether or not they are assembled. Total protein levels therefore report little; what reports is localisation into discrete structures, which is an imaging measurement.

Where adhesion dynamics is the question, image it. Where composition is the question, a blot of an enriched fraction is informative, provided the enrichment is shown to have worked.

Common questions

Why does my matrix protein not appear on a blot?

Because it was not solubilised. Cross linked matrix stays in the pellet through standard lysis, and the sample that matters is the one most people discard. Sequential or denaturing extraction is required.

Do junction proteins need retrieval?

Usually, in cross linked fixed tissue, and the optimal conditions differ between reagents. A weak junction stain is far more often a retrieval problem than a reagent problem.

How do I know the matrix signal is from my cells?

Run the coating and the medium without cells through the same protocol. Serum and coatings contribute matrix proteins, and that background is frequently substantial in culture experiments.

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