Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma, Taufkirchen, Germany). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in PBS. The following antibodies were used: alpha-Tubulin (Sigma, Taufkirchen, Germany), HOXB5 (Santa Cruz Biotechnology, Heidelberg, Germany), and BCL6 (Cell Signaling Technology, Danvers, MA, USA). For loading control, blots were reversibly stained with Poinceau (Sigma, Taufkirchen, Germany) and detection of alpha-Tubulin (TUBA) performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western Lightning ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
Poinceau
Manufactured by Merck Group
Sourced in Germany
Poinceau is a laboratory equipment used for the detection and visualization of proteins in electrophoresis gels. It is a staining solution that binds to basic amino acid residues, producing a red-pink color. The intensity of the color is proportional to the amount of protein present, allowing for the quantification of protein content.
Automatically generated - may contain errors
Lab products found in correlation
Western lightning ecl, by PerkinElmer (14 mentions)
Sigmafast protease inhibitor cocktail, by Merck Group (14 mentions)
α tubulin, by Merck Group (14 mentions)
Nitrocellulose membrane, by Bio-Rad (14 mentions)
Chemostar imager, by Intas (14 mentions)
Pvdf membrane, by Cytiva (3 mentions)
Gradient gel, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Nkx2 2, by Aviva Systems Biology (2 mentions)
Erk sc 94, by Santa Cruz Biotechnology (2 mentions)
Eef2k, by Cell Signaling Technology (2 mentions)
Hrp conjugated secondary goat antibodies, by Agilent Technologies (2 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
Cyclin d3, by Cell Signaling Technology (2 mentions)
Cyclin d2, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Hoxb5, by Santa Cruz Biotechnology (1 mentions)
Pdgfrb, by R&D Systems (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
Phospho stat5, by Cell Signaling Technology (1 mentions)
17 protocols using poinceau
1
Standardized Western Blotting Protocol
2
Western Blot Analysis of Signaling Proteins
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Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: MSX1 (R & D Systems), alpha-Tubulin (Sigma), PDGFRB (R & D Systems), phospho-PDGFRB (Aviva Systems Biology, Eching, Germany), NKX2-2 (Aviva Systems Biology) and PITX1 (Abnova, Taipei, Taiwan). For loading control blots were reversibly stained with Poinceau (Sigma) and detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany). PDGFD and BMP4 were quantified in the medium by ELISA using according Quantikine ELISA kits from R & D Systems. Samples were obtained by harvesting supernatants of 1x106 cells which were washed in PBS and subsequently incubated in 1 ml medium for 24 h.
3
Western Blot Analysis of IRF4 Expression
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Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma) and IRF4 (NOVUS Biologicals, Colorado, USA). For loading control blots were reversibly stained with Poinceau (Sigma) and detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
4
Protein expression analysis by Western blot
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Western blots were generated by the semi-dry method. Proteins obtained from cell line lysates using SIGMAFast protease inhibitor cocktail (Sigma) were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), AUTS2 (Origene), SMAD1 (Santa Cruz Biotechnology, Heidelberg, Germany), STAT5 (Santa Cruz Biotechnology), phospho-STAT5 (Cell Signaling Technology, Danvers, MA, USA). For loading control the blots were reversibly stained with Poinceau (Sigma) and then detection of alpha-Tubulin (TUBA) or SMAD1 was performed. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
5
Western Blot Analysis and FGF2 ELISA
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Western blots were generated by the semi-dry method. Proteins obtained from cell line lysates using SIGMAFast protease inhibitor cocktail (Sigma) were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), OTX1 (Santa Cruz Biotechnology, Heidelberg, Germany), OTX2 (Abcam, Cambridge, UK), and ZHX1 (Santa Cruz Biotechnology). For loading control the blots were stained with Poinceau (Sigma) and then detection of alpha-Tubulin (TUBA) was performed. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
Quantification of FGF2 protein in cell culture supernatants was performed as follows: log-phase cells were washed twice in PBS and cultivated in fresh medium at 1x106 cells per ml and supernatants harvested after 24 h. ELISA was performed using the Human FGF basic Quantikine ELISA Kit (R&D Systems) and an ELISA reader (Thermo Electron, Vantaa, Finland).
Quantification of FGF2 protein in cell culture supernatants was performed as follows: log-phase cells were washed twice in PBS and cultivated in fresh medium at 1x106 cells per ml and supernatants harvested after 24 h. ELISA was performed using the Human FGF basic Quantikine ELISA Kit (R&D Systems) and an ELISA reader (Thermo Electron, Vantaa, Finland).
6
Western Blot Protein Detection Protocol
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Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (T6199, Sigma), HMX2 (NBP1-91997, Novus Biologicals, Abingdon, UK), HMX3 (LS-B8011, LSBio, Eching, Germany), ERK (sc-94, Santa Cruz Biotechnology, Heidelberg, Germany), phosphor (P)-ERK (sc-7383, Santa Cruz Biotechnology), EPX (LS-C354350, LSBio), HTR7 (LS-C358892, LSBio). For loading controls blots were reversibly stained with Poinceau (Sigma) and detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
7
Biotinylation and Streptavidin Isolation
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Confluent cells grown on poly-d-lysine (Sigma-Aldrich) coated 10cm plates were labeled with 0.13 mg/mL sulfo-NHS-SS- biotin in PBS, during 1h on rocking table in +4C, after two washes of PBS cells were scraped off and sonicated in 500 μL PBS (supplemented with protease inhibitors). To 450ul 50ul of streptavidin coated beads (New England Biolabs inc.) was added, and 50 μL was analyzed as whole cell lysate. Beads and protein suspension was incubated retaining in +4C overnight, to be analyzed with western blot.
Nuclear fraction was isolated and protein concentrations were determined using a BCA Protein Assay Kit (Pierce). Equal amounts of cellular protein (30 μg) were separated on a 4%–12% gradient gel (Novex) and subsequently transferred to a PVDF membrane (Amersham). Total protein was visualized with Poinceau (Sigma). After blocking the membranes in TBS containing 5% BSA (Sigma), 0.02% Triton X-100 for 1 h, primary antibodies were added: After washing, the appropriate DyLight 800 or 580 fluorescently-conjugated secondary goat antibodies (Thermo Scientific, 1:10 000) were added in block solution for 1 h in room temperature on a rocking table. Antibody binding was detected using the Li-COR Odyssey CLx. Primary antibody (see resource table) incubations were carried out at 4°C overnight. All primary antibodies (resource table) were used (1:1000) for WB applications.
Nuclear fraction was isolated and protein concentrations were determined using a BCA Protein Assay Kit (Pierce). Equal amounts of cellular protein (30 μg) were separated on a 4%–12% gradient gel (Novex) and subsequently transferred to a PVDF membrane (Amersham). Total protein was visualized with Poinceau (Sigma). After blocking the membranes in TBS containing 5% BSA (Sigma), 0.02% Triton X-100 for 1 h, primary antibodies were added: After washing, the appropriate DyLight 800 or 580 fluorescently-conjugated secondary goat antibodies (Thermo Scientific, 1:10 000) were added in block solution for 1 h in room temperature on a rocking table. Antibody binding was detected using the Li-COR Odyssey CLx. Primary antibody (see resource table) incubations were carried out at 4°C overnight. All primary antibodies (resource table) were used (1:1000) for WB applications.
8
Western Blotting and IL-17F ELISA Protocol
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Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (T6199, Sigma), HLX (Novus Biologicals, Abingdon, UK), STAT3 (9139, Cell Signalling, Leiden, Netherlands) and phospho-STAT3 (9136, Cell Signalling). For loading controls blots were reversibly stained with Poinceau (Sigma) and detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
IL17F protein was quantified in cell culture supernatants using the IL-17 Human ELISA kit (Thermo Fisher Scientific). To harvest the supernatants 1 × 106 cells were washed in PBS and cultured in 1 ml fresh medium. After 24 hours supernatants were centrifuged and stored at –20°C. To optimize the range of the kit supernatants were diluted 1:50 and 1:100 in PBS.
IL17F protein was quantified in cell culture supernatants using the IL-17 Human ELISA kit (Thermo Fisher Scientific). To harvest the supernatants 1 × 106 cells were washed in PBS and cultured in 1 ml fresh medium. After 24 hours supernatants were centrifuged and stored at –20°C. To optimize the range of the kit supernatants were diluted 1:50 and 1:100 in PBS.
9
Western Blotting and BMP2 ELISA Protocol
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Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma, Taufkirchen, Germany). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma, #T6199), IRX1 (Biozol, Eching, Germany, #DF3225), and IRX3 (Biozol, #MBS8223417). For loading control, blots were reversibly stained with Poinceau (Sigma) and the detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
The enzyme-linked immunosorbent assay (ELISA) was used to quantify BMP2 protein levels in the supernatant of cell cultures. In addition, 2 × 106 cells were cultured in 2 mL of fresh medium in a 24-well plate. After 24 h, 1 of mL medium was harvested and frozen in aliquots. Quantification was performed using the Quantikine ELISA BMP-2 kit (R & D Systems, #DBP200), as described by the company. Two biological replicates were analyzed in triplicate.
The enzyme-linked immunosorbent assay (ELISA) was used to quantify BMP2 protein levels in the supernatant of cell cultures. In addition, 2 × 106 cells were cultured in 2 mL of fresh medium in a 24-well plate. After 24 h, 1 of mL medium was harvested and frozen in aliquots. Quantification was performed using the Quantikine ELISA BMP-2 kit (R & D Systems, #DBP200), as described by the company. Two biological replicates were analyzed in triplicate.
10
Western Blot Analysis of NKX2-3 Protein
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Western blots were generated by the semi-dry method. Protein lysate from human spleen was obtained from Origene. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma, Taufkirchen, Germany). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma) and NKX2-3 (Abcam, Cambridge, UK). For loading control blots were reversibly stained with Poinceau (Sigma) and detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
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