Figure 1: Structure of the Collagen II target protein.

Collagen II Target Introduction

Protein Function

• Collagen II is a fibrillar collagen protein composed of three identical α1 chains and is the primary collagen protein synthesized by chondrocytes.
• Collagen II is crucial for the normal development of embryonic skeletal, linear growth, and the ability of cartilage to resist pressure.
• Collagen II is typically covalently cross-linked with collagen IX and interacts with proteoglycans rich in leucine, providing tissue stability and strength and allowing for integrity and resistance to compression.
• Mutations in Collagen II can lead to cartilage dysplasia and premature osteoarthritis.

Protein Expression

• Human Collagen II isoform 2 is highly expressed in immature chondrocytes.

Protein Localization

• Secreted protein is secreted into extracellular space and extracellular matrix.

Isoforms & Post-Translational Modifications

• Human (P02458):
  Isoforms P02458-1~P02458-2: 134.3-141.7 kDa (predicted)
  Isoform P02458-3: 29.7 kDa (predicted)
• Mouse (P28481):
  Isoforms P28481-1~P28481-7: 131.8-141.9 kDa (predicted)
• Rat (P05539): 134.5 kDa (predicted)
• Post-translational modifications include hydroxylation and O-glycosylation.

WB Experiment Tips

Precautions

• Collagen II is expressed explicitly in cartilage tissue, so preparing high-quality target proteins from cartilage tissue/cells can help solve the problem of no signal or weak signal. For more detailed extraction protocols of Collagen II, please refer to reference ^[4].
• If antibodies developed through undenatured three-dimensional epitopes (such as ab34712) are used, it is necessary to avoid collagen denaturation during the experiment.
• Due to the instability of collagen compared to other proteins, detection should be performed at 4°C/on ice.
• To prevent collagen aggregation, pay attention to the experimental reagents' pH value, temperature, and collagen concentration. Collagen is soluble in acidic solutions, so an acidic environment (e.g., 0.5 M acetic acid, maintained at pH 2.5 for 24 hours) is essential for the stability and solubility of collagen. In alkaline pH, collagen will aggregate and form a gel. If the concentration of collagen is too high, it will also lead to gel formation.
• Optimize electrophoresis conditions by adding detergents, reducing agents, and denaturing agents to the samples. A 6% acrylamide gel can be used for Collagen II electrophoresis.
• Adding 4 M urea to the samples can improve the separation of collagen during electrophoresis.
• We recommend using natural protein standards as positive and negative controls.

Positive control

• Rat articular cartilage lysate

Example of results

Figure 2: WB experiment results of Collagen II protein, using recombinant Anti-Collagen II antibody [EPR27418-25] (ab307674).

Lane 1: Rat articular cartilage tissue lysate

Loading amount: 20 µg
Primary antibody dilution ratio: 1/1000
Predicted band size: 141 kDa
Observed band sizes: 110, 142 kDa

The bands at 142 kDa and 110 kDa represent full-length Collagen II and Collagen alpha-1(II) chain, respectively.

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex protease inhibitor to avoid target protein degradation.
2. Select a suitable lysis buffer to enrich more target proteins.
3. Keep the sample on ice throughout the sample preparation process.
4. Determine the protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.
2. For target proteins with larger molecular weight (such as molecular weight> 100 kDa), use a lower concentration separation gel for electrophoresis.
3. It is recommended to use positive and negative controls.

Transfer:

1. For target proteins with larger molecular weight, it is recommended to use a 0.45 μm PVDF membrane.
2. We recommend using 10% methanol or a lower concentration in the transfer buffer for target proteins with larger molecular weight.
3. After the PVDF membrane is activated, wash it thoroughly to remove residual methanol on the membrane completely.
4. We recommend using Ponceau S staining after transfer to determine if the transfer is successful (if fluorescence labeling detection is selected, make sure Ponceau S is completely washed off).

Blocking:

1. There is no blocking solution suitable for all systems. Please choose the appropriate blocking solution.

IHC experiment tips

Precautions

• Due to the high content of glycosaminoglycans and proteoglycans in cartilage tissue, this may mask the antigenic epitopes of Collagen II. If weak or no signal is observed when using cartilage tissue for IHC detection, consider performing enzyme antigen retrieval on the sections, such as incubation with 0.1% papain and/or 0.1% trypsin ^[5].
• When detecting Collagen II by IHC, if highly mineralized bone tissue samples, such as tibia and knee joint tissues, are selected, please note that decalcification is required after fixation to meet the subsequent staining requirements. The decalcification time and choice of decalcifying agent usually need to be optimized based on tissue size, type, and bone density. The degree of decalcification of the samples needs to be continuously monitored during the decalcification process ^[6].

Positive control

• Rat articular cartilage tissue

Example of results

Figure 3: IHC experimental results of Collagen II protein, recombinant Anti-Collagen II antibody [EPR27418-25] (ab307674).

Sample name: Rat articular cartilage tissue section
Primary antibody: diluted 200 times
Antigen retrieval method: heat-induced antigen retrieval, Tris/EDTA buffer (pH 9.0)

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample fixation:

1. The fixation time of the sample depends on the size of the tissue block and the type of tissue, but for most samples, such as fixation with 4% PFA, fixing at room temperature for 18-24 hours is more appropriate.
2. Insufficient fixation can result in a higher signal at the edges of the sample, a weaker signal at the center, or even no signal.
3. Excessive fixation can block antigenic epitopes. Although antigen retrieval can expose some of the epitopes, if the tissue fixation time is very long (such as more than a week), there may still be no signal after antigen retrieval.

Antigen retrieval:

1. We recommend using a pressure cooker for heat-induced antigen retrieval when performing immunohistochemistry experiments on paraffin sections. You can try fixing the sections at 110°C for 15 minutes. After retrieval, let it cool naturally to avoid putting it in cold water, which may cause detachment of the sections.
2. Microwave retrieval can also be chosen for tissues that are relatively brittle and prone to detachment during high-temperature and high-pressure retrieval, such as bone and cartilage, or tissues with small cutting surfaces, such as the sciatic nerve.

Blocking:

1. If using HRP conjugates for detection, please use 3% hydrogen peroxide to treat the sections for 10 minutes to block endogenous peroxidase.
2. If using fluorescently labeled secondary antibodies for the experiment, we recommend using a blocking solution containing 1% BSA and a final concentration of 0.3 M glycine to quench the autofluorescence caused by aldehyde groups.

References

1. M C Ryan, L J Sandell et al. Differential expression of a cysteine-rich domain in the amino-terminal propeptide of type II (cartilage) procollagen by alternative splicing of mRNA. J Biol Chem. 1990 Jun 25;265(18):10334-9.
2. K Gelse  1 , E Pöschl, T Aigner, Collagens--structure, function, and biosynthesis. Adv Drug Deliv Rev. 2003 Nov 28;55(12):1531-46. doi: 10.1016/j.addr.2003.08.002.
3. Peter Kannu, John Bateman, Ravi Savarirayan et al. Clinical phenotypes associated with type II collagen mutations. J Paediatr Child Health. 2012 Feb;48(2):E38-43. doi: 10.1111/j.1440-1754.2010.01979.x.
4. Z Deyl, I Miksík, A Eckhardt. Preparative procedures and purity assessment of collagen proteins. J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jun 25;790(1-2):245-75. doi: 10.1016/s1570-0232(03)00158-2.
5. Nerlich, A.G. (2003). Histochemical and Immunohistochemical Staining of Cartilage Sections. In: An, Y.H., Martin, K.L. (eds) Handbook of Histology Methods for Bone and Cartilage. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59259-417-7_22
6. Gayle M. Callis,18 - Bone, Editor(s): John D. Bancroft, Marilyn Gamble, Theory and Practice of Histological Techniques (Sixth Edition),Churchill Livingstone,2008,Pages: 333-363,https://doi.org/10.1016/B978-0-443-10279-0.50025-7.