Protocol tips for working with collagen

Collagen antibodies

​Collagens are highly conserved proteins, characterized by an uninterrupted glycine-X-Y triplet repeat that gives them their triple helical structure. It is the close similarity of this triple helical structure across the different collagen types that can make it very difficult to generate antibodies specific for a particular collagen.

In order to make a collagen antibody specific, the immunogen used for the antibody production must either be dependent on non-denatured three-dimensional collagen epitopes or shorter peptide sequences only found in specific collagens. Depending on the type of antibody you use, you will need to adapt your protocol accordingly. 

See below for protocols and tips for 

• Collagen extraction
• Antibodies recognizing non-denatured 3-dimensional collagen
• Antibodies recognizing short peptide sequences​
• Select the right antibody

Figure 1. Formalin-fixed paraffin-embedded sections of human colon tissue staining collagen I with ab138492 (top left). Immunofluorescence analysis of Human articular chondrocytes, staining Collagen II with ab34712 (top right). Ab21286 staining Collagen I (red) in Mouse colon tumor cells by ICC (bottom left). Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Collagen VI with ab182744 (bottom right). 

Collagen extraction

There are three main types of collagen extraction commonly used.

1. Salt precipitation extraction – used for saline soluble collagen
   Extraction in neutral salt solutions by the gradual addition of sodium chloride.
   eg  0.45M NaCl at pH 7.5 for 24 hours with stirring
2. Acid extraction – used for acid soluble collagen
   Extraction in an organic acid such as acetic acid 
   eg  0.5M acetic acid at pH 2.5 for 24 hours
3. Enzyme extraction – used for pepsin soluble collagen
   Extraction in an organic acid with the addition of pepsin. 
   eg  0.1% (w/v) pepsin in 0.5M acetic acid at pH 2.5 for 48 hours

​Figure 2. Comparative data of the extracted soluble collagen fractions. ²Adapted from Mocan E.Tagadiuc O.Nacu V. (2011) Aspects of collagen isolation procedure. Clin. Res. Studies 2, 3–5

Extraction by salt precipitation yields low levels of collagen (Figure 2). Therefore, either acid or enzyme treatment is usually preferred to isolate collagen with pepsin being the most efficient method as it can remove the non-helical ends of collagen and increases collagen solubility. However, extracted collagen can be partially digested as pepsin can cleave the terminal non-helical regions that contain the intermolecular cross-linkages.

The unwinding of the triple helices is temperature sensitive. Therefore, continuous refrigeration throughout collagen extraction is important to avoid degradation and denaturation.
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Protocol tips for antibodies recognizing non-denatured 3D collagen

If you are using collagen antibodies which are developed using non-denatured 3D epitopes (e.g. ab34710) you must be careful not to denature the collagen protein during your experiment. There will be diminished reactivity of your antibodies with denatured collagen or formalin-fixed, paraffin embedded tissues. It will state on the individual antibody datasheet if your antibody recognizes non-denatured 3D collagen, so be sure to check this first. Here you can find some advice on how to adapt your protocols to take this into account. 

ELISA
Use collagen standards with native 3D structures.
Due to the high sequence similarity of collagens, only the tertiary structure is different enough to distinguish between the different collagen types. Therefore, collagen-specific antibodies rely on the presence of the native structure for specific detection and collagen standards with native 3D structures should be used.

Sandwich ELISA is recommended.
For collagen antibodies which specifically recognize non-denatured three-dimensional epitopes, a sandwich ELISA is recommended as this will maintain the native collagen structure.

Indirect ELISA is not recommended because the native structure of collagen is disrupted when directly bound to the ELISA plate and therefore the specificity of the antibody may be lost.

Western blot
Take care with pH, temperature, and concentration to avoid collagen polymerization. 
​Collagen is soluble in acidic conditions so an acidic pH is critical for collagen stability and solubility. At a basic pH, collagen will polymerize and form a hydrogel. This will also happen if too high concentrations of collagens are used. Equally, as collagens are more unstable than other proteins, assays should be performed at <10⁰C.

Optimize gel running conditions
> Add detergents, reducing and chaotropic agents to samples.
> Use a 6% acrylamide gel for collagens I, II, and III.
> Use a 10% acrylamide gel for collagens IV, V, and VI. Alternatively, a gradient gel may be more suitable to see the full-length and smaller fragments.

Note: We recommend using native protein standards as positive and negative controls.

Recommended antibody dilution: 0.5-1ug/ml.

Immunohistochemistry - formalin fixed paraffin embedded
Determine the extent of cross-linking and choose suitable antigen retrieval conditions
​Crosslinking in collagens can occur where the collagen triple helices line up to form fibrils and then fibers. The degree of cross-linking can vary and will determine the antigen retrieval conditions needed to expose the epitope. Picrosirius red (ab150681) specifically binds to the [gly-X-Y]n helical structure on fibrillar collagen (type I–V) and therefore can be used to determine the extent of collagen cross-linking.

Where possible, harsh antigen retrieval conditions should be minimized to not disrupt the tertiary structure of collagen. We recommend antigen retrieval with sodium citrate buffer, pH 6.0, however enzymatic digestion is also commonly used.

Always follow the recommended antigen retrieval method indicated on the datasheet.

Recommended antibody dilutions: 1:500-1:2000

Protocol tips for antibodies recognizing short peptide sequences

If you are using collagen antibodies which are developed using short collagen peptides (e.g. ab138492 ) you do not need to be so careful to maintain the collagen 3D structure. All of our RabMAb^® collagen antibodies are made using a short synthetic peptide which is sequence specific to the collagen of interest. This allows you to use your normal western blot, ELISA, or   IHC protocols with no adaptation to take into account. For further tips and protocols, you can check our protocols book here which includes protocols for ELISA, IHC/ICC, and western blot. 

Vertebrate collagen distribution

Table 1. Vertebrate collagens. ¹Adapted from Shoulders MD, Raines RT. COLLAGEN STRUCTURE AND STABILITY. Annual review of biochemistry. 2009;78:929-958. doi:10.1146/annurev.biochem.77.032207.120833.

References
1. Shoulders MD, Raines RT. COLLAGEN STRUCTURE AND STABILITY. Annual review of biochemistry. 2009;78:929-958. doi:10.1146/annurev.biochem.77.032207.120833.
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2. Mocan E.Tagadiuc O.Nacu V. (2011) Aspects of collagen isolation procedure. Clin. Res. Studies 2, 3–5