The assay was performed as follows25 (link). Briefly, the purified proteins were used to perform the pull-down assay in a reaction system comprising 800 µL PBS buffer, 5 µM (final concentration) MBP-RpfG and HtsH1C-Flag-His, HtsH2C-HA-His or HtsH3C-Myc-His proteins, and 50 µL Dextrin Sepharose High Performance (Sigma-Aldrich, St. Louis, MO, USA). All samples were incubated at 4 °C overnight. The agarose was collected by centrifugation and washed ten times with PBS containing 1% Triton X-100 to remove non-specifically bound proteins. The MBP-bead-captured proteins were eluted by boiling in 6× SDS loading dye for 10 min. These samples were subjected to SDS-PAGE and Western blotting. Protein detection involved the use of MBP-specific (ab49923), Flag-specific (ab1162), HA-specific (ab187915), Myc-specific (ab32072), and His-specific (ab18184) antibodies obtained from Abcam, UK.
Ab49923
Manufactured by Abcam
Sourced in United Kingdom
Ab49923 is a laboratory product manufactured by Abcam. It is a piece of lab equipment designed for general use in scientific research. The core function of this product is to [product description]. No further details or interpretations about the intended use of this product can be provided.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ab49923
1
Protein-Protein Interaction Pulldown Assay
2
Pull-down Assay for Protein Interactions
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The assay was performed as described previously 24 . Brie y, the puri ed proteins were used to perform the pull-down assay in a reaction system comprising 800 µL PBS buffer, 5 µM ( nal concentration) MBP-RpfG and HtsH1C-Flag-His, HtsH2C-HA-His or HtsH3C-Myc-His proteins, and 50 µL Dextrin Sepharose High Performance agarose (Sigma-Aldrich, St. Louis, MO, USA). All samples were incubated at 4°C overnight. The agarose was collected by centrifugation and washed 10 times with PBS containing 1% Triton X-100 to remove non-speci cally bound proteins. The MBP-bead-captured proteins were eluted by boiling in 6× SDS loading dye for 10 min. These samples were subjected to SDS-PAGE and Western blotting. Protein detection involved the use of MBP-(ab49923), Flag-(ab1162), HA-(ab187915), Myc-(ab32072), and His-(ab18184) speci c antibodies obtained from Abcam, UK.
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