Figure 1: Structure of Collagen I target protein.

Collagen I Target Introduction

Protein Function

• Collagen I, a fibrous collagen protein, is the most abundant and essential protein in the human body, especially for the skin, bones, and connective tissues.
• Well-folded Collagen I is a structure of heterotrimer formed by intertwining two α1 chains and one α2 chain. Collagen trimers have high tensile strength.
• After secretion into the extracellular matrix, Collagen I undergoes proteolytic cleavage to form mature collagen protein. Mature collagen protein can self-assemble into highly ordered collagen fibers.
• Collagen I undergoes post-translational modifications, some of which are involved in collagen fiber synthesis. In contrast, others, such as isomerization, racemization, glycosylation, and other modifications, can change the biological function of Collagen I. The end products have been proven to be biomarkers for some diseases.
• Mutations in Collagen I are also associated with some skeletal and connective tissue diseases, such as osteogenesis imperfecta and Ehlers-Danlos syndrome.

Protein Expression

• Expressed in almost all connective tissues.
• Main protein in bones, skin, tendons, ligaments, sclera, cornea, and blood vessels.

Protein Localization

• Extracellular matrix.

Image 2: ICC experimental result of Collagen I protein, using Anti-Collagen I antibody (ab138492). Green: Collagen I, Red: alpha Tubulin, Blue: DAPI.

Isoforms & post-translational modifications

• Human (P02452): 138 kDa (predicted)
• Rat (P02454): 138 kDa (predicted)
• Mouse (P11087): 138 kDa (isoform 1); 118 kDa (isoform 2) (predicted)
• Hydroxylation
• O-glycosylation

WB Experiment Tips

Precautions

• Multiple bands of Collagen I may be observed during detection, such as the 220 kDa collagen I precursor protein, 60-75 kDa cleavage fragments, and 35 kDa C-peptide cleavage bodies, as shown in Figure 3. Please note that these observed bands may vary when detecting different types of samples or using different antibodies.
• Collagen I exhibits tissue-specific expression, with relatively weak detection signals in heart, kidney, and lung tissues (Figure 4). If the expression level of the target protein in the sample is uncertain, we recommend choosing skin tissue as a positive control. Optimizing experimental conditions, such as increasing antibody concentration, sample loading, and extending exposure time, can also enhance the detection signal.
• Collagen I has many cleavage sites in the sample, so degradation or cleavage can quickly occur if the sample is not handled correctly. Therefore, we recommend using freshly cultured collected cells to prepare the sample and increasing the concentration of protease inhibitors in the lysis buffer to avoid protein degradation as much as possible.
• Due to the instability of collagen proteins compared to other proteins, detection should be performed at 4°C/on ice.
• To prevent collagen protein aggregation, please pay attention to the experimental reagents' pH value, temperature, and collagen protein concentration. Collagen protein is soluble in acidic solutions, so an acidic environment (such as 0.5 M acetic acid, maintained at pH 2.5 for 24 hours) is crucial for the stability and solubility of collagen protein. In alkaline pH, collagen protein will aggregate and form a gel. If the concentration of collagen protein used is too high, gel formation can also occur.
• Optimize electrophoresis conditions: add detergents, reducing agents, and denaturing agents to the sample. 6% acrylamide gel can be used for Collagen I electrophoresis.
• Adding 4 M urea to the sample can improve the separation of collagen protein during electrophoresis.
• We recommend using natural protein standards as positive and negative controls.
• In WB experiments, multiple bands may be observed due to the immunogen of some antibody products (such as ab260043) being recombinant fragments that also contain glycosylation modification sites.

Positive control

• Human skin tissue lysate

Negative control

• HT-29 whole cell lysate

Figure 3: Recombinant Anti-Collagen I antibody [RM1131] (ab316222).

Lane 1: HFF-1 whole cell lysate.
Lane 2: HT-29 whole cell lysate.
Lane 3: NIH/3T3 whole cell lysate.
Lane 4: NIH/3T3 culture supernatant.
Band sizes observed: 220, 37, 60-75 kDa.

Observed molecular weights consistent with literature descriptions (PMID: 23940311; PMID: 9512508).

Figure 4: Western blot results of Collagen I protein, Anti-Collagen I antibody [EPR24331-53] (ab270993).

Lane 1: Mouse skin tissue lysate.
Lane 2: Mouse kidney tissue lysate.
Lane 3: Mouse lung tissue lysate.
Lane 4: Mouse heart tissue lysate.

Predicted band size: 139 kDa
Detected band size: 139 kDa

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 sufficient amount of composite protease inhibitor to avoid degradation of the target protein.
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 total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20μg total protein for electrophoresis.

Transfer:

1. For target proteins with larger molecular weight, it is recommended to add SDS to a final concentration of 0.1% in the transfer buffer.
2. For target proteins with larger molecular weight, it is recommended to use a PVDF membrane with a pore size of 0.45 μm.
3. For target proteins with larger molecular weight, it is recommended to use 10% methanol or lower concentration in the transfer buffer.
4. We recommended using Ponceau S staining after transfer to confirm the success of the transfer (if using fluorescence labeling detection, make sure the Ponceau S is completely washed off).

Blocking:

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

References

1. Christopher Niyibizi, David R. Eyre. Structural characteristics of cross-linking sites in type V collagen of bones. Chain specificities and heterotypic links to type I collagen. Eur J Biochem (1994).224:943-950. doi:10.1111/j.1432-1033.1994.00943.x.
2. Mitsuo Yamauchi, Marnisa Sricholpech. Lysine post-translational modifications of collagen. Essays Biochem (2012).52:113–133. doi: 10.1042/bse0520113.
3. Stéphanie Viguet-Carrin, Patrick Garnero, PD Delmas. The role of collagen in bone strength. Osteoporos Int (2006).17:319–336. doi:10.1007/s00198-005-2035-9.
4. DJ Leeming, Kim Henriksen, Inger Byrjalsen et al. Is bone quality associated with collagen age? Osteoporos Int (2009).20(9): 1461–1470. doi: 10.1007/s00198-009-0904-3.
5. Claudia Broder, Philipp Arnold, Sandrine Vadon-Le Goff. Metalloproteases meprin α and meprin β are C- and N-procollagen proteinases important for collagen assembly and tensile strength. Proc Natl Acad Sci U S A (2013). 110(35): 14219-14224. doi: 10.1073/pnas.1305464110.
6. Miriam T Levy, Maria Trojanowska, Adrian Reuben. Oncostatin M: a cytokine upregulated in human cirrhosis, increases collagen production by human hepatic stellate cells (2000).32(2): 218-226. doi: 10.1016/s0168-8278(00)80066-5.