Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.
Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.
Mammary gland growth and development occurs mainly after birth. This organ constantly remodels itself throughout the reproductive life of a mammal1. The adult mammary gland epithelium is comprised of an inner layer of luminal epithelial cells and an outer layer of basal cells, mainly composed of myoepithelial cells, surrounded by a basement membrane2. For a good review on mammary gland structure and development, the reader can refer to Sternlicht1. Cell-cell interactions via gap (GJ), tight (TJ), and adherens (AJ) junctions are necessary for the normal development and function of the gland1,3,4,5,6. The main components of these junctions in the murine mammary gland are Cx26, Cx30, Cx32, and Cx43 (GJ); Claudin-1, -3, -4, and -7 and ZO-1 (TJ); and E-cadherin, P-cadherin, and β-catenin (AJ)7,8. The levels of expression of these different junctional proteins vary in a stage-dependent manner during mammary gland development, suggesting differential cell-cell interaction requirements9. GJ, TJ, and AJ are linked structurally and functionally and tether other structural or regulatory proteins to the neighboring sites of adjacent cells, thus creating a junctional nexus10. The composition of the junctional nexus can impact bridging with the underlying cytoskeleton, as well as nexus permeability and stability, and can consequently influence the function of the gland8,9,10,11. The components of intercellular junctions residing in junctional nexuses or interacting with one another at different developmental stages of mammary gland development were analyzed recently using co-immunofluorescence (co-IF) and co-immunoprecipitation (co-IP)9. While other techniques allow for the evaluation of the functional association between proteins, these methods are not presented in this manuscript.
As proteins merely act alone to function, studying protein-protein interactions (PPIs), such as signal transductions and biochemical cascades, is essential to many researchers and can provide significant information about the function of proteins. Co-IF and microscopic analysis help to evaluate a few proteins that share the same subcellular space. However, the number of targets is limited by the antibodies, which must be raised in different animals, and by the access to a confocal microscope equipped with different wavelength lasers and a spectral detector for multiplexing. Co-IP confirms or reveals high-affinity physical interactions between two or more proteins residing within a protein complex. Despite the development of novel techniques, such as fluorescence resonance energy transfer (FRET)12 and proximity ligation assay (PLA)13, which can simultaneously detect the localization and interactions of proteins, co-IP remains an appropriate and affordable technique to study interactions between endogenous proteins.
The step-by-step method described in this manuscript will facilitate the study of protein localization and PPIs and point out pitfalls to avoid when studying endogenous PPIs in the mammary glands. The methodology starts with the presentation of the different preservation procedures for the tissues required for each technique. Part 1 presents how to study protein co-localization in three steps: i) the sectioning of mammary glands, ii) the double- or triple-labeling of different proteins using the co-IF technique, and iii) the imaging of protein localization. Part 2 shows how to precipitate an endogenous protein and identify its interacting proteins in three steps: i) lysate preparation, ii) indirect protein immunoprecipitation, and iii) binding partner identification by SDS-PAGE and Western blot. Each step of this protocol is optimized for rodent mammary gland tissues and generates high-quality, specific, and reproducible results. This protocol can also be used as a starting point for PPI studies in other tissues or cell lines.
All animal protocols used in this study were approved by the University Animal Care Committee (INRS-Institut Armand-Frappier, Laval, Canada).
1. Identifying Protein Co-localization
2. Studying PPIs
NOTE: Abdominal mammary glands should be used to study PPIs, as thoracic glands are in close association with the pectoral muscles. Excise the mammary glands (for a complete description of this procedure, refer to Plante et al.)14 and keep them at -80 °C for later use.
To determine whether GJ, AJ, and TJ components can interact together in the mammary gland, co-IF assays were first performed. Cx26, a GJ protein, and β-Catenin, an AJ protein, were probed with specific antibodies and revealed using fluorophore-conjugated mouse-647 (green, pseudocolor) and goat-568 (red) antibodies, respectively (Figure 1B and C). Data showed that they co-localize at the cell membrane of epithelial cells in the mice mammary gland on lactation day 7 (L7), as reflected by the yellow color (Figure 1D). Secondly, Claudin-7, a TJ protein; E-cadherin, an AJ protein; and Connexin26 (Cx26), a GJ protein, were probed with their specific antibodies and were revealed with fluorophore-conjugated rabbit-488 (green), mouse-555 (red), and mouse-647 (coral blue; pseudocolor) antibodies, respectively (Figure 2B-D). E-cadherin and Claudin-7 co-localization is displayed as a yellow-to-light-orange color, while Cx26 co-localization with E-cadherin and Claudin-7 resulted in white punctuated staining in mice mammary glands on pregnancy day 18 (P18) (Figure 2E).
To find out which junctional proteins intermingle and physically tether together at the cell membrane, co-immunoprecipitation was performed using mammary gland tissues from lactating mice (L14). Results showed that Cx43, a component of GJ, interacts with E-Cadherin and Claudin-7, but not with Claudin-3 (Figure 3A and B). These results were confirmed by the reciprocal IP; when E-Cadherin was immunoprecipitated, it interacted with Cx43 and Claudin-7 (Figure 3C).
Figure 1: β-Catenin and Cx26 co-localize at the cell membrane in mice mammary glands. Cryosections from mammary glands of mice at lactation day 7 (L7) were cut (7 µm) and processed for immunofluorescent staining. (A) Nuclei were stained with DAPI (blue). (B) Cx26 (green, pseudocolor) and (C) β-Catenin (red) are shown combined with appropriate fluorophore-labeled antibodies. (D) A merged image. Images were obtained with a confocal microscope equipped with a spectral detector. DAPI was visualized using the following settings: emission wavelength, 450.0 nm; excitation wavelength, 402.9 nm; laser power, 1.2; detector gain, PMT HV 100; PMT offset, 0. Cx26 (647) was visualized using the following settings: emission wavelength, 700.0 nm; excitation wavelength, 637.8 nm; laser power, 2.1; detector gain, PMT HV 110; PMT offset, 0. β-Catenin (568) was visualized using the following settings: emission wavelength, 595.0 nm; excitation wavelength, 561.6 nm; laser power, 2.1; detector gain, PMT HV 110; PMT offset, 0. Scale bars = 50 µm. Please click here to view a larger version of this figure.
Figure 2: Connexin26 (Cx26), E-cadherin, and Claudin-7 co-localize at the cell membrane in mice mammary glands. Cryosections from mammary glands on pregnancy day 18 (P18) were cut (7 µm) and processed for immunofluorescent staining using (B) Claudin-7 (green), (C) E-Cadherin (red), and (D) Cx26 (coral blue; pseudocolor), combined with the appropriate fluorophore-labeled antibodies. (A) Nuclei were stained with DAPI (blue). Images were obtained with a confocal microscope equipped with a spectral detector. DAPI was visualized using the following settings: emission wavelength, 450.0 nm; excitation wavelength, 402.9 nm; laser power, 5.4; detector gain, PMT HV 65; PMT offset, 0. Claudin-7 (488) was visualized using the following settings: emission wavelength, 525.0 nm; excitation wavelength, 489.1 nm; laser power, 5.0; detector gain, PMT HV 12; PMT offset, 0. E-Cadherin (568) was visualized using the following settings: emission wavelength, 595.0 nm; excitation wavelength, 561.6 nm; laser power, 13.5; detector gain, PMT HV 45; PMT offset, 0. Cx26 (647) was visualized using the following settings: emission wavelength, 700.0; excitation wavelength, 637.8; laser power, 5.0; detector gain, PMT HV 55; PMT offset, 0. Scale bars = 50 µm. Please click here to view a larger version of this figure.
Figure 3: Cx43, E-Cadherin, and Claudin-7, but not Claudin-3, are involved in a protein complex. Cx43 (A and B) and E-Cadherin (C) were immunoprecipitated using 500 mg of total lysates from mammary glands of mice at lactation. IPs and lysates were loaded in gels and transferred on PVDF membranes. Because Claudin-7 and Claudin-3 have the same molecular weight, they could not be analyzed on the same membrane. Thus, two parallel IPs were performed with the same lysate for Cx43, loaded on two gels, and transferred (membranes A and B). Membrane A was first probed with Cx43 to confirm the efficiency of the IP (top panel). Then, the membrane was sequentially probed with E-Cadherin and Claudin-7. Western blot analysis showed that the two proteins interact with Cx43. Membrane B was first probed with Cx43 to confirm the efficiency of the IP (top panel) and then probed with Claudin-3. Western blot analysis showed that Claudin-3 did not IP with Cx43, demonstrating that the two proteins do not interact. Membrane C was first probed with E-cadherin to confirm the efficiency of the IP (top panel). Then, the membrane, was sequentially probed with Cx43 and Claudin-7. Western blot analysis confirmed the interactions between the proteins. Please click here to view a larger version of this figure.
Cell-cell interactions via junctions are required for the proper function and development of many organs, such as the mammary gland. Studies have shown that junctional proteins can regulate the function and stability of one another and activate signal transduction by tethering each other at the cell membrane10. The protocols presented in the current manuscript have provided interesting findings about junctional protein differential expression, localization, and interaction during normal murine gland development9. Given that junctional protein localization is critical to the function of the proteins, and because they are known to interact with scaffolding proteins and numerous kinases3, co-IF and co-IP are effective techniques in the field of cell-cell interactions. Not only are these methods essential to enlighten the necessity of junctional nexuses in mammary gland development and their dysregulation in breast cancer, but they can also be used in other tissues and in experiments using cell lines.
The mammary gland is composed of two main compartments: the stroma and epithelium4. The adult epithelium is made of two layers of cells. In this approach, the proximity of the potential binding partners was determined using the co-IF technique, and their physical interactions were confirmed using co-IP. Co-IF has been successfully used by others to demonstrate the co-localization of proteins within the same tissue, structure, cell, or intracellular compartment17,18. The main advantage of this technique is the visual information it brings about the cellular or subcellular localization of each protein within the different cell types composing the tissue. Although this technique is quite simple, some recommendations must be followed. For instance, to avoid tissues damage, always add the solutions one drop at a time using a 200 µL pipette or a transfer pipette. This will allow for the immersion of the tissue surface without damaging the tissues. Similarly, remove the solutions using a Pasteur pipette placed beside the tissues by gently tilting the slide. Moreover, for antibodies whose storage solution contains glycerol, careful suction of the wash buffers is required to reduce the background. Moreover, the presence of milk proteins, such as caseins, during lactation can interfere with the antibodies by trapping them, resulting in false positives. A critical analysis of the resulting image is thus required, specifically at this stage.
This co-IF technique also has certain limitations or pitfalls. First, it requires specific antibodies. As mentioned previously (step 1.1), it is recommended to use one section (i.e., the one on the right side) on each slide for staining following the steps described above, adding the primary and secondary antibodies sequentially. For the remaining section (i.e., the one on the left side), follow the same procedure, using TBS-polysorbate 20 0.1% instead of the primary antibody solutions. While it is also possible, and even better, to verify the specific binding of the antibody using peptide competition, peptides used to generate commercial antibodies are not always available. It is thus important to verify the specificity of the binding using positive and negative controls (i.e., tissues or cells known to express, or not, the protein of interest). Moreover, antigen fixation sites can be inaccessible, especially for formalin-fixed tissues, thus resulting in the absence of a specific signal. An antigen-retrieval procedure may thus be required for some tissues. A short incubation with detergent can also be performed prior to antigen-retrieval to permeabilize the cell membrane. Second, for multiplexing, antibodies must be raised in different animals. For instance, if anti-rabbit was used in step 1.2.6, anti-mouse could be selected in step 1.2.10, but not another antibody raised in rabbit. Since most commercial antibodies are raised in rabbits, mice, or goats, it is sometimes difficult, or even impossible, to target two proteins at the same time due to the lack of appropriate antibodies. To overcome these limitations, one can either buy commercially available, pre-labeled antibodies or label primary antibodies with fluorophores using commercially available kits. Third, another shortcoming is linked to the excitation and emission of the different fluorophores. To avoid overlap of the signals from two different antibodies, the excitation-emission spectrum of each fluorophore must be separated. Thus, the number of targets that can be analyzed at once will vary according to the configuration of the available microscope. Finally, the quality of the analysis is highly dependent upon the microscope used. More detailed and precise data can be obtained using a confocal microscope compared to an epifluorescent microscope. The use of super-resolution microscopy can reveal protein co-localization in even more detail19.
Although co-IF brings important insights about the proximity of potential binding partners, it should be complemented by other methods to identify physical interactions between proteins. Among the available methods, co-IP is probably one of the most affordable to perform, as the equipment and material are easily accessible. Using an antibody bound to magnetic beads, one can isolate protein complexes and identify the components present in that complex using typical Western blot analysis. Similar to co-IF, some recommendations should be followed for best practices. For instance, it is recommended to minimize the samples when homogenizing the tissues to reduce the time between steps 2.1.5 and 2.1.9. While an experienced person can process up to 10 samples at a time, a beginner should not handle more than 4-6 samples. Similarly, the number of tubes should be limited when performing the IP protocol for the first time. It is recommended to start with a negative control (i.e., IgG) and a positive control (i.e., a tissue known to contain the protein to be immunoprecipitated) only. The second trial should be dedicated to optimization (see step 2.2.2). Once these steps give satisfying results, samples to be analyzed can be processed. Note that a negative control should always be included in the procedure.
This co-IP technique also has potential pitfalls. First, it requires tissues to be homogenized in conditions permitting the preservation of the links between proteins. For membrane proteins, such as junctional proteins, it is also crucial to use a buffer that will preserve the bonds between the proteins while also allowing their solubility. Second, similar to co-IF, it requires high-affinity antibodies for both the target protein and the binding partners. Moreover, because proteins remain in their tertiary conformation and protein complexes are not dissociated by homogenization, if the antibody recognizes part of the target protein that is in close proximity to the binding domain of a partner or that is hidden inside the native structure of the protein, the IP can be compromised. It is thus essential to always verify the efficiency of the IP using Western blotting before concluding the absence of a binding partner. Third, co-IP can generate false-positive results because of the protein either binding directly to the beads or precipitating during the procedure without being part of the complex. To identify these artifacts, an IgG control is required, as well as a reciprocal IP, as described in the methods presented. It is also possible to add a "pre-cleaning" procedure between steps 2.2.2 and 2.2.3 to avoid the unspecific binding of proteins to the beads. To do so, incubate the lysates with 50 µg of beads for 1 h at 4 °C on a roller mixer. Remove the beads with the magnetic stand and proceed to step 2.2.3. Fourth, the heavy and light chains of IgG can be the same size as the proteins of interest or the binding partner, thereby masking the signal. One solution is to dissociate the IgG chains with glycine, as described in this manuscript. It is also possible to purchase secondary antibodies that only recognize native antibodies and therefore do not bind to the denatured light and heavy chains loaded in the membrane (see the Table of Materials). The two methods may sometimes have to be combined. Fifth, co-IP allows for the identification of a limited number of binding partners, in part because of the number of antibodies that can be probed on the membrane. It also requires the pre-identification of these binding partners, either by co-IF or through a literature review. Finally, co-IP allows for the identification of the proteins present within a complex, and not for the direct interaction between two proteins.
Results from co-IP can be analyzed in different ways. It is possible to solely identify interacting partners by re-probing the same membrane with different antibodies, as described in this manuscript. It is also possible to quantify this interaction. To do so, the amount of the protein immunoprecipitated is first quantified by probing the membrane with an antibody against this protein. The signal intensity is analyzed using an imaging software, as described in this manuscript. Then, the membrane is re-probed with an antibody against the binding partner, and the signal intensity also quantified. The interactions between the two proteins can then be expressed as a ratio of the amount of the binding partner to the amount of the immunoprecipitated protein. However, to allow for comparison, the different samples must be processed at the same time and loaded on the same membrane. Biological replicates can be proceeded and analyzed similarly, and statistical analysis can be performed.
In the last few years, other techniques were developed to analyze PPIs. For instance, FRET allows for the identification of interacting proteins through energy transfer from one tag to another, only when the proteins are close enough to interact20. However, because it requires proteins to be tagged, this technique cannot be used in tissues. Similarly, it is possible to identify PPIs using a protein fused with a bacterial biotin ligase, BirA21. This ligase will add biotin to proteins that come in close proximity (i.e., interact) with the chimeric protein. While this method is innovative and unbiased, it cannot be performed in tissues. Alternatively, PLA can be used in tissues. Similar to co-IF, this assay is based on antibody affinity. For this assay, secondary antibodies are tagged with DNA sequences that can interact when they are in close proximity (i.e., upon PPIs) and form a circular DNA molecule22. This circular DNA molecule is then amplified and detected using fluorescently labeled complementary oligonucleotides. Although this assay is elegant, it requires many validation steps and still relies on primary antibody affinity and some knowledge of potential interacting partners. Finally, an unbiased alternative to the traditional IP assay has also been developed to identify PPIs. In rapid immunoprecipitation-mass spectrometry of endogenous protein (RIME) assays, IP samples are analyzed by mass spectrometry (IP/MS)23 instead of Western blot analysis. The main advantage of this high-throughput method is that it provides massive information about all the endogenous interacting proteins using few materials. However, it requires access to a peptide-sequencing instrument23.
It is important to mention that, after several preliminary tests, each step of this protocol has been optimized for the mammary gland. However, the method can surely be used for other organs after a few modifications. For co-IF, the optimal temperature for mammary gland sectioning, suitable blocking and washing and solutions, and the proper antibody concentrations were all tested. For co-IP, various lysis and elution buffers and methods of extractions were also tested and have a major impact on IP success. In sum, each step of this protocol is important to obtaining high-quality and reproducible results with the least possible background and the most specificity. While other methods are available, co-IF followed by co-IP remain valid and simple methods to evaluate PPIs. These two techniques can be used both in tissues and in cell lines and only require a few validation steps and controls.
The authors have nothing to disclose.
I.P. is funded by a Natural Sciences and Engineering Research Council of Canada grant (NSERC #418233-2012); a Fonds de Recherche du Québec-Santé (FRQS), a Quebec Breast Cancer Foundation career award, and a Leader Founds grant from the Canadian Foundation for Innovation grant. E.D. received a scholarship from the Fondation universitaire Armand-Frappier.
Mice strain and stage | St. Constant, Quebec, Canada | C57BL/6 Femals; pregnancy day 18 (P18) and lactation day 14 (L14), Charles River Canada | |
PBS 10X (stock) | 1) Dissolve 80g NaCl (F.W.: 58.44), 2g KCl (F.W. 74.55), 26.8g Na2HPO4•7H2O (F.W. 268.07) and 2.4g KH2PO4 (F.W.:136.09) in 800ml distilled water. 2) Adjust the PH to 7.4 3) Add water to reach to the 1 litre final volume. |
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TBS 10X (stock) | 1) Dissolve 60.5g TRIS, 87.6g NaCl in 800ml distilled water. 2) Adjust the PH to 7.5 3) Add water to reach to the 1 litre final volume. |
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Name | Company | Catalog Number | Comments |
Part 1-Immunofluorescence | |||
Freezing media | VWR International, Ville Mont-Royal, QC, Canada | 95067-840 | VMR frozen sections compound |
Microtome | Mississauga, ON, Canada | 956640 | Microm HM525, Thermo fisher scientific HM525 NX Cryostat 115V 60Hz |
Blades | C.L. Sturkey, Inc. Les Produits Scientifiques ESBE St-Laurent, QC, Canada | BLM1001C | High profile gold coated blades |
Pap pen | Cedarlane, Burlington, ON, Canada | 8899 | Super PAP Pen, Thermo fisher scientific |
Microscopic slides | Fisher Scientific, Burlington, ON, Canada | 12-550-15 | Fisherbrand Superfrost Plus Microscope Slides |
Formaldehyde | BioShop Canada Inc, Burlington, ON, Canada | FOR201.1 | Forlmadehyde |
Bovine Serum Albumin (BSA) | Santa Cruz Biotechnology, Inc, California, USA | ||
Blocking solution | 3% BSA in TBS | ||
Wash solution | TBS-Tween 20 0.1% | ||
Polysorbate 20 | Oakville, ON, Canada | P 9416 | Tween 20, Sigma-Aldrich |
Mounting media | Cedarlane, Burlington, ON | 17984-25(EM) | Fluoromount-G |
First & secondary antibodies | Cell Signaling, Beverly, MA, USA | Mentioned in Column D | E-Cadherin (4A2) Mouse mAb (#14472s) 1/50 (Cell Signaling) with anti-mouse IgG Fab2 Alexa Fluor 555 (#4409s), Cell Signaling |
First & secondary antibodies | Life technologies, Waltham, MA, USA & Cell Signaling, Beverly, MA, USA | Mentioned in Column D | Claudin-7 (#34-9100) 1/100 (Life Technologies) with anti-rabbit IgG Fab2 Alexa Fluor 488 (#4412s) (Cell Signaling) |
First & secondary antibodies | Santa Cruz Biotechnology, Inc, California, USA; Fischer Scientific, Burlington, ON, Canada | Mentioned in Column D | β-Catenin Antibody (C-18): sc-1496 (SANTA CRUZ) with anti-Goat IgG (H+L) Alexa Fluor 568 (#A11057), Molecular Probe (Fisher Scientific) |
First & secondary antibodies | Life technologies, Waltham, MA, USA & Cell Signaling, Beverly, MA, USA | Mentioned in Column D | Connexin26 (#33-5800) 1/75 (Life Technologies) with anti-mouse IgG Fab2 Alexa Fluor 647 (#4410s) |
Nuclei stain | Fisher Scientific, Burlington, ON, Canada | D1306 | DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) 1/1000 in PBS |
Fluorescent microscope | Nikon Canada, Mississauga, On, Canada | Nikon A1R+ confocal microscopic laser equipped with a spectral detector | |
Software of IF images analysis | Nikon Canada, Mississauga, On, Canada | NIS-elements software (version 4) | |
Name | Company | Catalog Number | Comments |
Part 2-Immunoprecipitation | |||
Triple-detergent Lysis buffer (100ml) pH=8.0 | 1) Mix 50mM TRIS (F.W. :121.14), 150mM NaCl (F.W. :58.44), 0.02% Sodium Azide, 0.1% SDS, 1% NONIdET P40, 0.5% Sodium Deoxycholate in 80ml distilled H2O. 2) Adjust the PH to 8.0 with HCl 6N (~0.5ml). 3) Adjust the volume to 100ml. Keep it in fridge. At the day of protein extraction, use 1/100 NaVo3, 1/100 protease/phosphatase inhibitor and 1/25 NAF in calculated amount of Triple detergent lysis buffer: Sodium Fluorid (stock) solution 1.25M (F.W. 41.98), Sodium Orthovanadate (stock) Solution 1M (F.W.: 183.9) |
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Protease/phosphatase inhibitor | Fisher Scientific, Burlington, ON | 78441 | Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X) |
Protein dosage | Thermo Scientific, Rockford, Illinois, USA | 23225 | Pierce BCA protein assay kit |
Tissue grinder | Fisher Scientific, Burlington, ON | FTH-115 | Power 125, Model FTH-115 |
Magnetic beads and stand | Millipore, Etobicoke, ON, Canada | PureProteome Protein G Magnetic Bead System (LSKMAGG02) | |
Wash solution for IP | PBS or PBS-Tween20 0.1% depending to the step | ||
Primary antibodies for immunoprecipitation | Cell Signaling, Beverly, MA, USA | Mentioned in coulmn D | IgG Rabbit (rabbit (DA1E) mAb IgG Isotype control (#3900s) (Cell Signaling) 0.5 µl/200 µl |
Primary antibodies for immunoprecipitation | Cell Signaling, Beverly, MA, USA | Mentioned in coulmn D | IgG Mouse mouse (G3A1) mAb IgG Isotype control (#5415s) (Cell Signaling) 0.5 µl/200 µl |
Primary antibodies for immunoprecipitation | Sigma-Aldrich, Oakville, ON, Canada | Mentioned in coulmn D | Connexin43 (#C6219) (Sigma-Aldrich) 4 µl/200 µl |
Primary antibodies for immunoprecipitation | Cell Signaling, Beverly, MA, USA | Mentioned in coulmn D | E-cadherin (4A2) Mouse mAb (#14472s) (Cell Signaling) 1 µl/200 µl |
Laemmli buffer | BIO-RAD, Mississauga, Ontario, Canada | 1610747 | 4x Laemmli Sample Buffer (Add β-mercaptoethanol following manufacturer recommendation) |
Acidic glycine | Fisher Scientific, Burlington, ON | PB381-5 | 0.2 M glycine; adjust pH=2.5 with HCl |
Tris | Fisher Scientific, Burlington, ON | BP152-1 | 1 M (pH=8) |
SDS-PAGE acrylamide gels | BIO-RAD, Mississauga, ON, Canada | 1610180 -5 | TGX Stain-Free FastCast Acrylamide Solutionss (7.8%, 10%, 12%) |
Running buffer 10x | BIO-RAD, Mississauga, ON, Canada | 1704272 | Tris 30.3g/glycine 144.1g /SDS 10g in 1 litre distilled water |
Membranes | BIO-RAD, Mississauga, ON, Canada | 1704272 | PVDF membranes, Trans-Blot Turbo RTA Mini PVDF Transfer Kit |
Transfer method | BIO-RAD, Mississauga, ON, Canada | 1704155 | Trans-Blot Turbo Transfer System |
Dry Milk | Smucker Food of Canada Co, Markham, ON, Canada | Fat Free Instant Skim Milk Powder, Carnation | |
Blocking solution for blots | 5% dry milk in TBS-Tween 20 0.1% | ||
Washing solutions for blots | TBS-Tween 20 0.1% | ||
Primary and secondary antibodies for blots (10ml) | Sigma-Aldrich, Oakville, Ontario & Abcam, Toronto, ON, Canada | Mentioned in column D | Connexin43 (#C6219) (Sigma-Aldrich) 1/2500 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5000 (Abcam, Toronto, ON, Canada) |
Primary and secondary antibodies for blots (10ml) | Cell Signaling, Beverly, MA, USA & Abcam, Toronto, ON, Canada | Mentioned in column D | E-cadherin (24E10) rabbit mAb 1/1000 (#3195s) (Cell Signaling) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5000 (Abcam, Toronto, ON, Canada) |
Primary and secondary antibodies for blots (10ml) | Life technologies, Waltham, MA, USA & Abcam, Toronto, ON, Canada | Mentioned in column D | Claudin-7 (#34-9100) (Life technologies) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5000 (Abcam, Toronto, ON, Canada) |
Primary and secondary antibodies for blots (10ml) | Life technologies, Waltham, MA, USA & Abcam, Toronto, ON, Canada | Mentioned in column D | Claudin3 (#34-1700) (Life technologies) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5000 (Abcam, Toronto, ON, Canada) |
Luminol solution for signal detection on blots | BIO-RAD, Mississauga, ON, Canada | 1705061 | Clarity Western ECL Blotting Substrate |
Imaging blots | BIO-RAD, Mississauga, ON, Canada | 1708280 | ChemiDoc MP imaging system |
Analayzing blots | BIO-RAD, Mississauga, ON, Canada | ImageLab 5.2 software |