Protein was extracted with SDS‐lysis buffer (0.5 mM EDTA, 20 mM HEPES, 2% (w/v) SDS pH 7.9), and protein concentrations were determined with the Bio‐Rad DC protein assay. Proteins were resolved by SDS–PAGE, transferred to PVDF membranes, and immunoblotted with primary and horseradish peroxidase‐conjugated secondary antibodies (Appendix Table S1 ). Active RAP1 pull‐down experiments were performed using the RAP1 Activation Assay kit (Abcam ab212011) according to manufacturer's instructions. The positive controls were included as shown in Source Data. Protein was visualized by Amersham enhanced chemiluminescence (ECL) Western Blotting Detection Reagent (GE Healthcare Life Sciences), and X‐ray film (Fujifilm SuperRX) or ChemiDoc™ Touch Imaging System (Bio‐Rad).
Superrx
Manufactured by Bio-Rad
SuperRX is a high-performance X-ray film designed for general radiographic and medical imaging applications. It provides consistent, high-quality imaging results with excellent clarity and contrast. The product specifications and technical details are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Tx 100, by Thermo Fisher Scientific (2 mentions)
Chemidoc, by Bio-Rad (2 mentions)
Amersham enhanced chemiluminescence, by GE Healthcare (1 mentions)
Rap1 activation assay kit, by Abcam (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
3 protocols using superrx
1
Protein Extraction and Western Blot Analysis
2
Affinity-based Protein Purification and Analysis
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Cells rinsed in phosphate-buffered saline (PBS) were mechanically lifted, harvested, and lysed in SLB + 1% LMNG or Triton X-100 (TX-100, Fischer Scientific), as described above. Lysates were clarified by centrifugation (17,000 x g, 30 min.) and pre-cleared using CL-4B Sepharose beads (50 µL of 50:50 slurry, Pharmacia/GE), with subsequent affinity- and immuno-purifications carried out using the resulting lysates. Beads were washed thrice in SLB and subsequently resuspended in 2 x Laemmli buffer + 20 mM DTT (10 min, 56°C) after the final wash, separated by SDS-PAGE and transferred to PVDF membrane for western blotting. Western blots were performed by incubating membranes in PBST blocking buffer (PBS + 1% Tween-20 supplemented with 5% non-fat dry milk), with subsequent primary and secondary antibody incubations in PBST + 5% non-fat dry milk. Secondary antibodies conjugated with horseradish peroxidase (HRP) were used to detect proteins bound to primary antibodies for enhanced chemiluminescence (ECL) with images captured either on X-ray film (FujiFilm, SuperRX) or by CCD camera (Chemidoc, BioRad) for quantification.
3
Immunoprecipitation and Western Blotting
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Cells rinsed in phosphate-buffered saline (PBS) were mechanically lifted, harvested, and lysed in SLB + 1% LMNG or Triton X-100 (TX-100, Fischer Scientific), as described above. Lysates were clarified by centrifugation (17,000 x g, 30 min.) and pre-cleared using CL-4B Sepharose beads (50µL of 50:50 slurry, Pharmacia/GE), with subsequent affinity and immunopurifications carried out using the resulting lysates. Beads were washed thrice in SLB and subsequently resuspended in 2 x Laemmli buffer + 20 mM DTT after the final wash, separated by SDS-PAGE and transferred to PVDF membrane for western blotting. Western blots were performed by incubating membranes in PBST blocking buffer (PBS + 1% Tween-20 supplemented with 5% non-fat dry milk), with subsequent primary and secondary antibody incubations in PBST + 5% non-fat dry milk. Secondary antibodies conjugated with horseradish peroxidase (HRP) were used to detect proteins bound to primary antibodies for enhanced chemiluminescence (ECL) with images captured either on X-ray film (FujiFilm, SuperRX) or by CCD camera (Chemidoc, BioRad) for quantification.
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