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Pull-down assay experiments using Tagged protein purification kits




Introduction of Pull-down assay experiments using Tagged protein purification kits

Proteins function by interacting with various other proteins. To reveal the function of a protein, it is important to identifiy other proteins which interact to the protein.

Protocol

Pulldown protocol

Experiment conditions

Pulldown condition

Exampe 1: Pull-down assay of an endogenous protein

 

p62/SQSTM1 directly interacts with LC3, which is localized on autophagosome membrane, and is degradated by autophagic-lysosome pathway. Interactions of p62/SQSTM1 with LC3 was confirmed by pull-down assay using the cell lysate of the HA-LC3 transfectant.

HA tagged Protein PURIFICATION KIT (MBL Code No. 3320)

Pulldown data1

Example 2: Pull-down assay of forcibly expressed proteins

 

Beclin 1, which is essential for early autophagosome formation, is reported to make complex with various proteins such as UBRAG and Vps34 and regulate the processes of autophagosome/lysosome fusion (Autophagosome maturation) and endocytosis. Using HA, c-Myc, and His-tagged protein purification kits, pull-down assay of Beclin1 and UVRAG is performed
*Samples: HA tagged Beclin1/Myc-His tagged UVRAG transfectant.

HA tagged Protein PURIFICATION KIT (MBL Code No. 3320)

Pulldown data2

c-Myc tagged Protein MILD PURIFICATION KIT Ver.2 (MBL Code No. 3305)

Pulldown data3

His tagged Protein PURIFICATION KIT (MBL Code No. 3310)

Pulldown data4