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Publication: Biology Open (2016) 0, 1-14 doi:10.1242/bio.014993
Application: Western Blot analysis,
Nkx2.5, a type of transcription factor, and desmin, a type of intermediate filament protein, are simultaneously expressed in cardiac progenitor cells during commitment of primitive mesoderm to the cardiomyogenic lineage. It is demonstrated that desmin can up-regulate Nkx2.5 expression, which suggests that desmin has the ability to influence cardiogenic commitment and myocardial differentiation through regulation of the nkx2.5 gene in cardiac progenitor cells. This experiment demonstrates that nkx2.5 reporter genes are activated by desmin, which rescues nkx2.5 haploinsufficiency in cardiac progenitor cells. Moreover, desmin is significant to adult cardiac side population stem cells due to its interaction with proper expression of Nkx2.5. These findings validate that desmin presents temporarily in the nuclei of differentiating cardiac progenitor cells and desmin interacts with the transcription factor to form complexes which bind to the promoter and enhancer at nkx2.5 gene. This research suggests that desmin plays a regulatory role in guiding cardiomyogenesis in cardiac stem cells.
In this experiment, at first, mCherry was used to confirm the nuclear localization of desmin without using desmin-specific antibodies. Researchers generated ESC and CVPC lines, expressing either a desmin-mCherry or a mCherry-desmin fusion protein, with the control being merely mCherry. mCherry-tagged desmin was expressed under the control of the CMV promoter in all cells. The mCherry-tagged desmin proteins succeeded in localizing to the nuclei, as found in the nuclei purified fractions of differentiating CPCs. Quantification of the nuclear fluorescence signals in differentiating CPCs suggests that only the fusion proteins could enter the nucleus. Researchers chose western blot analysis with mCherry specific antibodies and mCherry-tagged desmin proteins with chromatin to analyze the cytoplasmic, nucleoplasm, and chromatin fractions of mCherry-tagged desmin expressing CPCs (Fig. C). The same processing method used on murine heart cells, isolated 3 days post-partum demonstrated the nuclear localization of desmin in early postnatal heart cells (Fig. D). To validate the interaction of desmin with the regulatory elements of the nkx2.5 gene without using desmin-specific antibodies, ChIP analysis was performed in differentiating desmin-mCherry, and mCherry-desmin-expressing ESC- and CVPCs-derived CPCs with a monoclonal mCherry specific antibody.
Protocol: Isolation of nuclei and chromatin associated proteins and western blot analysis
To obtain cytoplasm-free nuclei from cells undergoing cardiomyogenesis a high-quality biochemical fractionation protocol (Rosner et al., 2013) was employed with some modifications allowing the separation of the nuclear membranes from the chromatin (Aaronson and Blobel, 1975). Cells from 6.7-day old EBs and 9.3-day old cardiac bodies, respectively, expressing either mCherry, the desmin-mCherry, or the mCherry-desmin fusion protein, were dissociated by incubation with a mixture of trypsin solution (Invitrogen, 27250018), with 0.1% (m/v) pancreatin (Sigma, P3292) and 0.05% (m/v) collagenase type 2 (Whortington, CLS-2) at 37°C for 25 min. Cells were suspended in M15, cell aggregates removed by low-speed centrifugation, and single cells were recovered by centrifugation. Cells were washed once with ice cold PBS and then carefully lysed in 5× the pellet volume of 20 mM Tris-HCl pH 7.6, 50 mM β-mercaptoethanol, 0.1 mM EDTA, 2 mM MgCl 2 , 174 µg/ml PMSF (Sigma, P7626), 2 µg/ml leupeptin (Sigma, L2884), 2 µg/ml aprotinin (Sigma, A6279), and 0.3 µg/ml benzamidine chloride (Sigma, B6506) (solution 1) at room temperature for 2 min and then at 4°C for 10 min. After adding 10% (v/v) of a 10% Nonidet-P40 (Sigma 74385) solution to the lysates, cell fragments were carefully suspended 3× with a 200 µl Gilson tip and nuclei were pelleted at 600×g and 4°C for 5 min. The supernatant containing the cytoplasmic fraction of the cells was removed and nuclei were washed to remove all cytoplasmic components from their surface. To maintain the integrity of the nuclei, they were carefully suspended 3× with a 200 µl Gilson tip in 0.5 ml of solution 1 containing 1% Nonidet-P40 and then pelleted at 600×g and 4°C for 5 min. This procedure was repeated twice and purity of nuclei was assessed by fluorescence microscopy. After removal of the last wash solution, nuclei were lysed in 15× the pellet volume of 6 mM NaH 2 PO 4 pH 7.1, 603 mM KCl, 171 mM NaCl, 1% Triton X-100, 8 mM β-mercaptoethanol, and 348 µg/ml PMSF in a micro douncer at 4°C for 1 h. Insoluble chromatin was pelleted at 17,000×g at 4°C for 25 min, the supernatant containing the karyoplasm was removed, and the chromatin solubilized in 1× SDS sample buffer (Laemmli, 1970) containing 1 mg/ml DNase I (Boehringer, 104159) by repeated pipetting at room temperature and final incubation at 96°C for 5 min. Samples of the cytoplasmic, karyoplasmic, and chromatin fraction were stored in 1× SDS sample buffer at −20°C.
The eukaryotic protein, Rad51, typically assists with the repair of double stranded DNA fractures, and is a major contributor in this process.
Our most recent review for this monoclonal primary antibody shows how the expression of Rad51 increases after DNA damage.
If you would like to try this antibody for yourself check out our Antibody Validation Project page to request a 20ul trial size sample.
Here's the image, but check out the full review here.
We had a number of requests to trial this beta-actin antibody, which has been validated for use in western blot.
Now we have received our second review, containing full protocol details and another 5-star rating.
If you would like to try this antibody please check out the Antibody Validation Project page, where you can request to sample a 20ul trial size for free.
Here's our most recent western blot result, in Human neuronal progenitor cells.
Check out the rest of the details here.
We followed up on our Immunofluoresence work to bring another element of validation to you. Now you can see the results we obtained from testing in Immunohistochemistry for 295 of our STJ9 model polyclonal and monoclonal antibodies - also covering 18 different tissues from 3 separate species.
The reports can be found on our slideshare page, or alternatively check the 'Slideshare Reviews' section of the individual product page.
We made sure that the images are large enough for you to see the finer details, but if you'd like to see even greater version quality please email us as firstname.lastname@example.org.
Check out our beta-actin antibody STJ91464, below.
Immunohistochemical analysis of paraffin embedded Mouse lung tissue. 1: Actin β Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only.
Immunohistochemical analysis of paraffin embedded Human colon tissue. 1: Actin β Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only.
And there's more where that came from.