The overall goal of the following experiment is to analyze protein stability in a cell-free system. This is achieved by inoculation of Oceana Biana with different agrobacterium strains. Harboring the protein of interest containing a binary construct.
The total protein is then extracted from the plant leaves. The leaves are transferred to micro fuge tubes and incubated at room temperature for increasing periods of time. Next protein samples are resolved by SDS Acrylamide, gel, electrophoresis, and electro transferred to a nitrocellulose membrane.
In order to detect the protein of interest using a specific antibody, results are obtained that show protein stability in the presence or absence of a specific proteasomal inhibitor based on western blotting analysis. We describe here a simple methodology to study the stability of the INE protosome cyst substrate in the presence or absence of one of the essential components of the proteasomal degradation pathway in a cell-free system. So these methods can provide insight into a safer proteasomal degradation.
It can also be applied to other system, such as studies of the roles of regulated protein degradation in diverse cellular processes. In this demonstration, NCOA hamana is used. The plant is easily grown, highly susceptible to agrobacterium and has large leaves which are easily inoculated.
Grow one plant for four to six weeks in a pot with promix B under environmentally controlled conditions of long day and 40 to 65%relative humidity. Fertilize the plant occasionally with commercially available products per the manufacturer's instructions. Once the plant is grown, select leaves with a size of 50 millimeters by 70 millimeters or larger.
For agrobacterium inoculation grow Agrobacterium strain harboring the binary construct, expressing the tested protein overnight at 28 degrees Celsius in YEP medium supplemented with appropriate antibiotics following centrifugation of the cells, resuspend to an optical density at 600 nanometers of 0.5 in infiltration buffer and incubate for two hours at room temperature. Infiltrate the culture into the AAL side of a leaf using a one milliliter needleless syringe. After infiltration, grow the plant for 72 hours under the same light regime Before harvesting, four hours before harvesting, infiltrate the agrobacterium inoculated leaf areas with 10 micromolar inhibitor or mock.
Treat the leaf with the respective solvents, harvest the inoculated areas of the leaf, usually 200 to 400 milligrams of fresh weight, and grind them into a fine powder in liquid nitrogen. Then prepare total protein extract by placing the ground tissue into 500 microliters of degradation buffer. Clarify the extract by two sequential centrifugations at 12, 000 times gravity for five minutes to perform the protein degradation reaction.
Transfer equal volumes of extracts, usually 20 microliters to micro fuge tubes and incubate them at room temperature for increasing periods of time. Typically, sample time zero as well as 5, 10, 15, 20, and 30 minute time points are used. Stop the reactions by boiling an SDS gel sample buffer before analyzing them by western blotting.
Resolve the protein samples by SDS poly acrylamide gel electrophoresis by first determining protein concentration using the Bradford Method and loading 50 to 80 micrograms of total protein per lane. Make sure that all samples are loaded equally for loading controls. Compare intensities of ubiquitous protein species such as putative rubis large chains, which migrate as a major band with a relative electrophoretic mobility of around 50 kilodaltons.
Following electro transfer of the resolved proteins to a nitrocellulose membrane according to standard protocols, block the membrane with 5%skim milk in TBST for one hour at room temperature. Next, dilute the primary anti epitope antibody in 1%Skim milk in TBST to the concentration recommended by the manufacturer. Incubate the antibody with the blocked membrane for one hour at room temperature or overnight at four degrees Celsius with gentle agitation.
Then rinse the membrane in 20 milliliters of TBST for 15 minutes once and then twice for five minutes at room temperature with gentle agitation. Next, dilute the secondary antibody conjugated with horse radish peroxidase in 1%Skim milk in TBST as recommended by the manufacturer. Incubate the secondary antibody with the membrane for one hour at room temperature with gentle agitation after another rinse in TBST, visualize the proteins of interest with a horse radish peroxidase representative experiments showing destabilization of a plant defense related protein.
Veep one by the VBFF box protein via the SCF pathway is demonstrated in N hamana. Western blood analysis demonstrated that Veep one amounts were reduced to a significant degree when co-expressed with VBF, but remained relatively stable in the absence of VBF co-expression. The proteasomal degradation mechanism of Veep one destabilization by VBF was inferred from its inhibition by proteasome inhibitor MG 1 32.
Quantification of these results demonstrated almost complete inhibition of V one by VBF, which was practically blocked by MG 1 32. The slightly higher levels of V one in the presence of MG 1 32, most likely are explained by inhibition of the endogenous VBF homolog activity. After washing this video, you should have a good understanding of how to study the protein degradation by the beginning proteasome pathway.
The same experimental design could be used for any other organisms and can be performed in order to answer additional questions like the roles of regulated protein degradation in diverse cellular processes.
