Total protein was extracted by RIPA lysis buffer (#P0013C, Beyotime Institute of Biotechnology, China) and quantified using a Pierce BCA Protein Assay Kit (#P0012S, Beyotime Institute of Biotechnology, China). The total protein was electrophoresed by 10% SDS-PAGE and transferred to PVDF membranes (#AR0136-04, BOSTER, China). The membranes were blocked in non-fat dry milk (~ 5%) for 60 min at room temperature and incubated with special primary antibodies at 4 °C overnight. After washing, the membranes were incubated with peroxidase-conjugated secondary antibody (anti-rabbit) at room temperature for 60 min. Protein expression levels were visualized using enhanced chemiluminescence kit (#P0020; Beyotime Institute of Biotechnology, China). Images were visualized with a ChemiDoc MP Imaging System (Bio-Rad, Hercules). Primary antibodies: EGR2 antibody, (#ab245228, dilution 1:1000, Abcam, UK); GAPDH antibody (#A19056, dilution 1:1000, ABclonal, China).
Egr2 antibody
Manufactured by Abcam
Sourced in United Kingdom
The EGR2 antibody is a tool used in research laboratories to detect and study the EGR2 (Early Growth Response 2) protein. EGR2 is a transcription factor that plays a role in the regulation of gene expression during cellular development and differentiation. This antibody can be used in various experimental techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to investigate the expression and localization of the EGR2 protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Donkey anti rabbit 555, by Thermo Fisher Scientific (2 mentions)
Bd554714, by BD (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Boster Bio (1 mentions)
Pierce bca protein assay kit, by Beyotime (1 mentions)
Gapdh antibody, by ABclonal (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
3 protocols using egr2 antibody
1
Western Blot Analysis of EGR2 Protein Expression
2
EGR2 Protein Detection in Fixed Cells
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Cells were fixed with Cytofix/Cytoperm Buffer (BD Biosciences, BD554714) for 10 min at room temperature. Cytofix/Cytoperm buffer was removed, and cells were washed twice with Hanks’ balanced salt solution containing 2% bovine serum albumin (BSA) and 1 mM EDTA. Cells were kept in permeabilization/wash buffer (BD Biosciences, BD554714) for 1 hour at 4°C or until the experiment was performed. Fixed cells were blocked using 3% BSA, 0.1% Triton–phosphate-buffered saline (PBS) for 30 min at room temperature and then with 1/200 of the EGR2 antibody (Abcam) overnight at 4°C. The next day, cells were washed with 0.1% Triton-PBS and incubated with 1/200 donkey anti-rabbit 555 (Thermo Fisher Scientific, no. A31572) secondary antibody and phalloidin (Abcam, ab176759) for staining actin filaments, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole. After washing with 0.1% Triton-PBS, slides were mounted with ProLong Gold Antifade Reagent (Life Technologies, no. 10144). Images were taken using a Leica SP8 with light deconvolution microscope.
3
Immunofluorescent Staining of EGR2 in Cells
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Cells were fixed with Cytofix/Cytoperm Buffer (BD, BD554714) for 10 min at room temperature.
Cytofix/Cytoperm buffer was removed and cells were washed twice with HBSS containing 2% BSA and 1mm EDTA. Cells were kept in permeabilization/wash buffer (BD, BD554714) for one hour at 4C or until the experiment was performed. Fixed cells were blocked using 3% BSA, 0.1% Triton-PBS for 30 min at room temperature and then with 1/200 of the EGR2 antibody (abcam) overnight at 4C. Next day, cells were washed with 0.1% Triton-PBS, incubated with 1/200 donkey anti-rabbit 555 (ThermoFisher, #A31572) secondary antibody, phalloidin (abcam, ab176759) for staining actin filaments and nuclei were counter-stained with DAPI. After washing with 0.1% Triton-PBS, slides were mounted with Prolong Gold Antifade Reagent (Life Technology, #10144). Images were taken using a Leica SP8 with light deconvolution microscope.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 3, 2020. ; https://doi.org/10.1101/2020.11.02.365742 doi: bioRxiv preprint
Cytofix/Cytoperm buffer was removed and cells were washed twice with HBSS containing 2% BSA and 1mm EDTA. Cells were kept in permeabilization/wash buffer (BD, BD554714) for one hour at 4C or until the experiment was performed. Fixed cells were blocked using 3% BSA, 0.1% Triton-PBS for 30 min at room temperature and then with 1/200 of the EGR2 antibody (abcam) overnight at 4C. Next day, cells were washed with 0.1% Triton-PBS, incubated with 1/200 donkey anti-rabbit 555 (ThermoFisher, #A31572) secondary antibody, phalloidin (abcam, ab176759) for staining actin filaments and nuclei were counter-stained with DAPI. After washing with 0.1% Triton-PBS, slides were mounted with Prolong Gold Antifade Reagent (Life Technology, #10144). Images were taken using a Leica SP8 with light deconvolution microscope.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 3, 2020. ; https://doi.org/10.1101/2020.11.02.365742 doi: bioRxiv preprint
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