Figure 1: Structure of the Occludin Target Protein.

Occludin Introduction

Protein Function

• Occludin is a membrane-integral protein that plays a role in the formation and regulation of tight junction (TJ) paracellular permeability barriers. Paracellular transport refers to the transport of molecules between adjacent epithelial cells, which tight junctions regulate to control paracellular permeability and maintain cell polarity.
• Occludin is able to induce adhesion when expressed in cells lacking tight junctions.
• Occludin act as a co-receptor for hepatitis C virus (HCV) in hepatocytes.

Protein Expression

• Occludin is expressed at tight junctions of epithelial cells and endothelial cells. It is highly expressed in the kidney but not detected in the testis.

Protein Characteristics

• Occludin has multiple isoforms, with molecular weights ranging significantly (8 kDa to 59 kDa).

Protein Localization

• Occludin is primarily localized to the cell membrane, cell junctions, or tight junctions.

Figure 2: Occludin ICC Experiment Results, Recombinant Anti-Occludin antibody [EPR20992] (ab216327).Green: Occludin; Red: Tubulin; Blue: DAPI

Isoforms & Post-Translational Modifications

• Human (Q16625): Isoforms 1-3: 54-59 kDa (predicted)
  Isoform 4: 32 kDa (predicted)
  Isoform 5: 23 kDa (predicted)
  Isoforms 6-7: 8 kDa (predicted)
• Mouse (Q61146): 59 kDa (predicted)
• Rat (Q6P6T5): 59 kDa (predicted); according to literature [5], multiple isoforms are also observed in rats.
• Occludin undergoes phosphorylation at different sites, which can regulate its interaction with other tight junction proteins such as ZO-1.
• Occludin may undergo protein cleavage (proteolysis).
• Occludin may undergo dimerization.
• Occludin may undergo ubiquitination.

WB Experiment Tips

Precautions

• Occludin exists in multiple isoforms with a wide range of molecular weights (8 kDa to 59 kDa), which may result in the detection of multiple bands in WB experiments.
• Expression of Occludin varies among different tissue cells, and some samples may show weak or no expression. Choose appropriate experimental samples and consider using positive controls to ensure the experimental system is functioning correctly.
• Occludin is a multi-pass transmembrane protein. High-temperature sample boiling can cause protein aggregation. It is recommended to use a milder boiling method. If no signal is detected, try skipping the boiling step.
• Occludin undergoes phosphorylation modifications, which may contribute to the detection of multiple bands in WB experiments.

Positive Control

• Caco-2 cells.

Negative Control (Low Expression)

• HeLa cells.

Example Results

Figure 3: Anti-Occludin Antibody (ab31721)

Lane 1: Wild-type HAP1 whole cell lysate at 40 µg
Lane 2: OCLN (Occludin) knockout HAP1 whole cell lysate at 40 µg
Lane 3: HeLa whole cell lysate (Low Occludin expression) at 20 µg
Lane 4: HepG2 whole cell lysate (High Occludin expression), 20 µg

Predicted Band Size: 59 kDa
Additional cross-reactive bands were observed in both wild-type and knockout cells

Figure 4: Recombinant Anti-Occludin Antibody [EPR20992] (ab216327)

Lane 1: Mouse colon tissue lysate
Lane 2: Mouse brain tissue lysate
Lane 3: Mouse lung tissue lysate
Lane 4: Rat colon tissue lysate
Lane 5: Rat brain tissue lysate
Lane 6: Rat lung tissue lysate

Predicted Band Size: 59 kDa
Actual Band Size: 65 kDa

Key control points

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

Sample preparation:

1. It is recommended to use freshly prepared samples, do not freeze, to prevent protein degradation.
2. It is recommended to use a milder boiling method.
3. Add a protease inhibitor cocktail to prevent degradation of target proteins.
4. Keep samples on ice throughout the entire sample preparation process.
5. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Transfer:

1. It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.

Antibody incubation:

1. Select a suitable antibody working concentration according to the product datasheet.

IHC Experiment Tips

Precautions

• Occludin expression varies among different tissues, and some samples may show weak or no expression. It is recommended to use positive controls to ensure the experimental system is functioning correctly.

Positive Control

• Kidney tissue

Figure 5: IHC with Recombinant Anti-Occludin Antibody [EPR20992] (ab216327)

Sample Name: Paraffin-embedded human colon tissue
Antigen Retrieval Method: Heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0.

Key control points

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

Sample fixation:

1. The optimal fixation time for samples depends on the size and type of tissue, but for most samples, it is advisable to fix with 4% PFA at room temperature for 18-24 hours.

Blocking:

1. When conducting experiments using secondary antibodies conjugated with fluorescent labels, it is recommended to add 1% BSA and a final concentration of 0.3M glycine to the blocking solution to quench the autofluorescence induced by aldehyde groups.
2. If using HRP conjugate for detection, treat the sections with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity.

Antigen retrieval:

1. When conducting immunohistochemistry experiments on paraffin sections, we recommend heat-induced antigen retrieval at 110°C for 15 minutes using a pressure cooker.

ICC Experiment Tips

Precautions

• Occludin is a multi-pass transmembrane protein, and the choice of fixative can significantly impact staining results. If the antigenic epitope is located on the inner side of the plasma membrane, or if cells are fixed with aldehydes (e.g., 4% PFA), permeabilization of cells is recommended.
• Due to Occludin's association with cell tight junctions, cell density is crucial for proper protein expression. Ensure appropriate cell density during cell experiments.
• The cellular state can affect proper protein localization. Therefore, before fixation, ensure that cells to be tested are in a normal state. Abnormal cell states may lead to mislocalization of the protein of interest.

Positive Control

• Caco-2 cells (human colorectal adenocarcinoma cell line).

Example Results

Figure 6: Recombinant Anti-Occludin Antibody [EPR20992] (ab216327)

Sample Name: Caco-2 cells

Experimental Method: Fixed with 4% PFA, permeabilized with 0.1% Triton X-100
Experimental Result: Confocal image showing membrane staining in the Caco-2 cell line

Key control points

In the experiment, in addition to paying attention to routine issues, it is also important to pay special attention to the following key control points:

Sample fixation:

1. Before starting, it is necessary to ensure cells are healthy and growing in a normal state. For the detection of Occludin, a confluency of 80% is suitable.

Permeabilization:

1. If using aldehydes (such as 4% PFA) for cell fixation, it is recommended to permeabilize with 0.1-0.25% Triton X-100 at room temperature for 10 minutes. Permeabilization is particularly critical if the target protein is located intracellularly.

Blocking:

1. It is recommended to add 0.3 M glycine to the blocking solution to quench the autofluorescence caused by aldehyde groups.

References

1. Gemma J Feldman, James M Mullin, Michael P Ryan. Occludin: structure, function and regulation. Adv Drug Deliv Rev. 2005 Apr 25;57(6):883-917. doi: 10.1016/j.addr.2005.01.009.
2. Alexander Ploss, Matthew J Evans, Valeriya A Gaysinskaya, Maryline Panis, Hana You, Ype P de Jong, Charles M Rice. Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature. 2009 Feb 12;457(7231):882-6. doi: 10.1038/nature07684. Epub 2009 Jan 28.
3. Takuya Suzuki, Bertha C Elias, Ankur Seth, Le Shen, Jerrold R Turner, Francesco Giorgianni, Dominic Desiderio, Ramareddy Guntaka, Radhakrishna Rao. PKC eta regulates occludin phosphorylation and epithelial tight junction integrity. Proc Natl Acad Sci U S A. 2009 Jan 6;106(1):61-6. doi: 10.1073/pnas.0802741106. Epub 2008 Dec 29.
4. Philip M Cummins. Occludin: one protein, many forms. Mol Cell Biol. 2012 Jan;32(2):242-50. doi: 10.1128/MCB.06029-11. Epub 2011 Nov 14.
5. Gwen McCaffrey, Melissa J Seelbach, William D Staatz, Nicole Nametz, Carolyn Quigley, Chris R Campos, Tracy A Brooks, Thomas P Davis. Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia. J Neurochem. 2008 Sep;106(6):2395-409. doi: 10.1111/j.1471-4159.2008.05582.x. Epub 2008 Jul 21.