Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model

Ubiquitination governed by the E one, E two, E three enzymatic cascade is a key regulator of protein function. Ubiquitin structural flexibility allows it to form diverse chains via different lysine or Met one linkages with each topology dictating a distinct outcome, K 48 for degradation, K 63 for signaling and repair and Met one for innate immunity. This study delineates and compares two different cell lysis methods to measure application levels in the mammalian cells using Smurf two, SMAD two as a model, which will assist researchers in selecting more appropriate methods to detect the ubiquitination level of substrate proteins.

Maintain HEK two 93 T cells in high-glucose DMEM supplemented with 10%FBS and one percent PS.Seed cells in six well plates at 500, 000 cells per well. Prepare transfection mixtures to represent 10%of the total culture value and use SMAD two ubiquitination as a experimental model. Combine different plasmids in 100 microliter of serum-free DMEM.

In a separate tube, dilute transfection reagent. As the reagent mixture dropwise to the plasmid solution, incubate for 15 minutes at room temperature and distribute evenly onto cells. Replace the transfection medium after six to hours with two microliter of fresh complete DMEM per well.

Treat cells with five nanometer MG one three two for 12 hours before harvesting. Aspirate medium and wash each well with one mil of prechilled PBS. Remove PBS completely.

Transfer the suspension to a one point five tube and place on ice. Centrifuge at three hundred and 80 XG for five minutes at four degrees. Remove the supernatant and retain the pellet.

Transfer 50 microliter of protein AG agarose beads per sample to a new tube. Wash beads three times with one mil of lysis buffer. Remove residual buffer without disturbing the pellet.

At 100 microliter of working solution and five microliter of antibody. Add 500 microliter of prechilled working solution to the pellet. Incubate on ice for 30 minutes.

Sonicate at two W using four secs on, four sec off cycles for five repetitions. Return samples to ice for the remainder of the incubation. Add 40 microliter of working solution to the pellet.

Add five microliter over 10%SDS while mixing continuously. Vortex briefly and heat at 95 degrees until the suspension becomes transparent and fully liquified. Invert intermittently to ensure uniform denaturation.

Add 450 microliter working solution. Sonicate at two W using four secs on four secs off cycles for five repetitions. Centrifuge lysates for 15 minutes at four degrees.

Reserve 60 microliter of lysate and add 15 microliter of five multi loading buffer. Heat at 95 degrees for 15 minutes and label as input. Add 100 microliter of antibody-coupled beads to the remaining lysate.

Incubate at four degrees for eight to 12 hours with rotation. Centrifuge beads at 100 XG for one minutes at four degrees and discard supernatant. Add 75 microliter of one multi SDS loading buffer and hit at 95 degrees for 15 minutes to elute proteins, label as IP.Detect ubiquinated proteins by western blot.

HE K two 93 T cells were transfected and stimulated with MG one 32. The cells were divided into five groups according to different treatment methods and the differences between each group are as follows, without flag SMAD two lane one, without MG one three two lane two, without FLAG antibody added into beads lane three, with MYC-SMURF two lane five. All cells were lysed using the ice-bath method.

Lane one served as a control to verify the successful over expression of the SMAD two plasmid. Lane two served as a control to demonstrated the inhibitory effect of MG one three two on the degradation of ubiquitinated proteins. Lane three served as a control to confirm that nonspecific binding has been completely eliminated.

Lane five was used to investigate the promoting effect of SMURF two on the ubiquitination of SMAD two. The greyscale value of HA in lane five was higher than that in lane four, which demonstrated that the SMURF two increased the ubiquitination level of SMAD two. When we lysecells using the heat treatment method we obtained the similar results as those with the ice-bath method, however, when using the heat treatment method, the expression of SMURF two could not be detected in the IP FLAG results.

We also demonstrated that the heat treatment method is also suitable for the detection of ubiquitination of endogenous proteins. We further compare the efficiency of the two methods in detecting ubiquitination levels whether in the input or IP flag results we found that the greyscale value of HA in the group using the heat treatment method was higher than that in the ice-bath method. To enhance signaling detection, we employed plasmid based over expression combined with MG one three two treatment during cell harvesting to prevent degradation of ubiquitinated proteins.

Lysate preparation requires careful modulation of na cl concentration as high salt disrupts weak interaction while low salt increases nonspecific aggregation. However, low concentrations may lead to excessive background from nonspecific aggregates. For enhanced protein enrichment recovery antibodies to beads at four degrees ensure more efficient capture of target proteins and reduces non-specific binding.

In heat treatment method, it's critical to ensure that concentration of SDS remains below one percent, access SDS may strip antibodies from beads. This study delineates and comparisons to different lysis methods to measure ubiquitination levels in mammalian cells using SMURF two SMAD two as a model which will assist researchers in selecting more appropriate methods to detect the ubiquitination levels of substrate proteins.