Tips for Collagen Antibodies

Production of antibodies capable of targeting type-specific collagens can present unique challenges for antigen design, screening strategies, and antibody validation especially if the goal is to generate an antibody capable of detecting collagen in its native, non-denatured form and recognize collagens in a type-specific manner. Denatured collagen and native collagen present immunologically distinct epitopes to the immune system for recognition. It is not possible to generate type-specific anti-collagen antibodies when denatured epitopes are used as immunogens because the features that make types of collagen distinct from one another are lost when the collagen is physically denatured.

Type-specific anti-collagen antibodies must first be affinity purified against the target type-specific collagen immobilized to beads, e.g. type I, and the resultant antibody must be sequentially cross-adsorbed against each of the other type-specific collagens, e.g. types II-VII, to render an antibody with the desired specificity. An additional challenge is to accomplish the purification and cross-adsorption steps while maintaining the immobilized collagen in its native form so that only antibodies recognizing collagen’s three-dimensional structure will be purified, which greatly enhances the antibody’s type-specific binding properties. Extreme pH, high salt concentration, and detergents must be avoided at all costs as these conditions will permanently disrupt the collagen structure. Nevertheless, highly functional anti-collagen antibodies can be used in assays like Western blotting when the recommended protocols are precisely followed by researchers.

Tips for Collagen Western Blotting

Collagens are insoluble in organic solvents, while water-soluble collagen represents only a small fraction of total collagen. Many procedures suggest the use of limited proteolytic digestion followed by salt extraction while maintaining the collagen preparation on ice. These suggestions are intended to minimize unwanted collagen degradation and denaturation.

Optimized protocols for detecting collagen by Western blotting include several steps that can be adjusted to generate the highest quality reproducible data. For instance, sample buffer for SDS-PAGE should be adjusted to contain 125 mM Tris-HCl, pH 6.8, 10% Glycerol, 5% SDS, and 0.007% bromide blue with or without 4% β-mercaptoethanol. Many researchers have achieved successful separation of collagens when 4M urea is included in the sample, which results in better separation of the collagens during migration through the running gel.

As collagen types, especially native collagen, display significant molecular size differences, selection of SDS-PAGE running gels with the proper percentage of acrylamide and cross-linking will generate better separation of collagen in gels. In general, 6% acrylamide gels work well for collagen types I, II, and III, while 10% acrylamide gels are recommended for collagen IV, V, and VI. More information on collagen protein separation from Rockland can be found on the Rockland website.

To minimize background staining and non-specific protein staining, we recommend the following tips:

• Use PVDF membrane and block the membrane with buffers containing 3% BSA.

  Note: Type-specific anti-collagen antibodies should be diluted from stock concentrations in blocking buffer to 1 µg/mL prior to use.

• After incubating the blot with primary antibody, wash the membrane with PBST (#MB-075-1000) for 15 minutes with gentle agitation at room temperature then decant and repeat the wash three additional times.

  Note: Never allow the blot to dry during processing.

• An optimized secondary antibody, like Rockland’s Anti-Rabbit IgG (H&L) Peroxidase Conjugated Pre-adsorbed (#611-103-122), is recommended for use at a 1:50,000 dilution from stock concentrations.

Tips for Enhanced Collagen ELISA Sensitivity

Sandwich ELISA techniques are recommended for collagen studies because this method can maintain the native structure of collagen as denaturing and dissociating conditions are not required. Sandwich or capture ELISA is more sensitive than direct or tittering ELISA methods. We recommend this straightforward and optimized ELISA protocol for high-quality results:

• Coating:

  • Coat the wells with 100 µL capture antibody at a concentration of 1–10 µg/mL
  • Cover the plate with adhesive plastic and incubate the plate overnight at 4°C
  • Decant the coating solution and wash with 250 µL of 1X PBST three times.

• Blocking:

  • Block the coated wells with 200 µL/well of blocking buffer containing 3% BSA
  • Cover the wells with adhesive plastic and incubate the plate at room temperature for 2 hours or at 4°C for overnight.

• Sample incubation:

  • Add 100 µL of collagen standards and test samples, incubate at 37°C for 60–90 minutes
  • Decant the sample, wash 3 times with PBST.

• Detection and secondary antibody incubation:

  • Add 100 µL of detection antibody, according to manufacturer’s optimal dilution
  • Cover the wells using adhesive plastic and incubate at room temperature for 2 hours
  • Wash the plate with PBST 3 times
  • Process for signal development and read plate.

Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) are recommended as detection enzymes due to their sensitivity and low levels of detection.

Tips for Collagen Immunohistochemistry

A comprehensive histological study of type-specific collagens assists in determining its distribution and abundance in each tissue sample. While many immunoassays designed to detect type-specific collagens can be challenging, including immunohistochemistry, high-quality and reproducible data can be generated when optimized protocols are used. As type-specific anti-collagen antibodies recognize native, non-denatured collagens, frozen section immunohistochemistry reliably produces excellent results.

Researchers have reported that the following recommendations for fixed tissues and cells will improve study outcomes:

• Fix tissue with formaldehyde, followed by blocking tissue with 2.5% horse serum for 1 hour at 25°C.

  Note: Certain tissues may require antigen retrieval methods and additional optimization.

• Both heat-induced epitope retrieval and protease-induced epitope retrieval can be used to unmask collagen epitopes and restore epitope-antibody binding.