Testing protein. Protein interaction is indispensable for dissection of protein functionality. This video introduces an in vitro protein protein binding assay to test the interaction between a membrane and immobilized protein with a soluble protein.
First, fusion proteins are cloned, expressed and extracted. The tagged insoluble protein is immobilized on nitrocellulose membrane and treated to refolded to its native confirmation. The membrane is then probed with tagged soluble proteins and immuno blotting is performed to detect the proteins.
If the tagged proteins interact, they're captured by the protein immobilized on the membrane, and the signal from the immuno blotting will be observed. Thus, this assay provides a reliable method to test interaction between an insoluble protein and a protein in solution. The main advantage of this technique over existing vessels like GST pull down, is that you can reliably assess the binding between insoluble protein and soluble protein in vitro.
The first step of this procedure is to generate genetically tagged proteins for detection. Begin by cloning the protein that will be immobilized onto the membrane. Refer to here as pro Im for immobilized protein into an expression vector, encoding a large tag such as GST, the empty expression vector, which codes only for the tag will be used as an immobilized protein negative control, and is referred to as pro IM nc.
Next, clone the protein that will be used as a soluble probe into an expression vector, encoding a different tag such as strep two. This will be referred to as prosol for soluble protein as a negative control, clone, a negative control soluble protein into the same expression vector. This will be referred to as Prosol NC for soluble protein negative control.
Next, use a protein expression system such as a coli or balo virus to express the tagged proteins the expression system used Should be carefully chosen based on the requirement for post-translational modifications. Extract the expressed proteins from the organism of choice. For pro IM and pro I mnc Resus.
Suspend the cells in SDS page loading buffer containing protease inhibitor cocktail. Then boil the cells for five minutes, then centrifuge them to remove the precipitate to extract Prosol and prosol NC.Use standard procedures. The pro IMS can be extracted under denaturing conditions because the proteins will be refolded after being immobilized in the nitrocellulose membrane.
However, prosol should be extracted to give a native confirmation because they will be added to the binding buffer once the tagged proteins have been extracted. Determine the concentration of the recombinant proteins using a standard method such as the BioRad protein assay kit. If crude extract rather than purified protein will be used for the assay.
Estimate the concentration of the protein of interest by scanning densitometry of the corresponding band on a kumasi stained SDS poly acrylamide gel. Using known concentrations of BSA as a reference. Then plot the standard curve based on the signal intensities measured by the scanning densitometry of the BSA and calculate the amount of protein contained in the crude extract.
The next step of the procedure is to immobilize pro Iam and pro IAM NC on the membrane. Begin by loading one microgram, each of pro Iam and pro IAM NC extracts in duplicate on an SDS poly acrylamide gel. After electrophoresis, remove the gel from the glass plates and place it in 100 milliliters of transfer buffer, then incubate with gentle agitation for 20 minutes at room temperature.
Following the incubation, perform a western blot to transfer the proteins to a nitrocellulose membrane after the transfer to allow the membrane bound proteins to refold. Place the membrane in 15 milliliters of buffer A and incubate it with gentle agitation. To remove residual SDS incubate for 15 minutes at room temperature with gentle agitation.
Following the incubation carefully remove the membrane from the buffer. Then place the membrane in 25 milliliters of denaturation buffer. Incubate for two hours at room temperature with gentle agitation.
During the incubation, the nitrocellulose membrane will become opaque. Next, using forceps, transfer the membrane to 25 milliliters of TBS and incubate with gentle agitation for five minutes. During this step, the membrane returns to its original white color.
Next, transfer the membrane to 25 milliliters of binding buffer. Incubate at four degrees Celsius with gentle agitation for overnight. Next, the soluble protein will be used to probe the membrane for the immobilized protein.
First, prepare the hybridization buffers dilute one to 10 micrograms of prosol and prosol NC in separate tubes containing 10 milliliters of fresh binding buffer. Then transfer the buffer to a hybridization bag. Next, cut the membrane into two strips, both containing pro IM and pro IAM nc.
These membranes will be placed in the hybridization buffer. Transfer each membrane into the prosol or prosol NC hybridization solution and incubate for 1.5 hours at room temperature with gentle agitation. Remove the membranes from the hybridization solutions and rinse three times for 15 minutes in TBS.
To visualize protein protein interactions, block the membrane with 2.5%skim milk in TBST for one hour. After the incubation, place the blocked membranes in the primary antibody solution. Here antis strep two polyclonal rabbit antibody is used.
Incubate for one hour at room temperature with gentle agitation following the incubation. Rinse the membranes in 20 milliliters of TBST for 15 minutes and twice for five minutes at room temperature with gentle agitation. Next place the membranes in the HRP conjugated secondary antibody solution Here, an anti rabbit IgG antibody conjugated to HRP is used.
Incubate for one hour at room temperature with gentle agitation. Rinse the membranes in 20 milliliters of TBST for 15 minutes and twice for five minutes at room temperature with gentle agitation as before, after the final rinse in TBS, visualize the protein protein interaction using an HRP chemiluminescent substrate Here Millipore. Im Molan Western Chemiluminescent.
HRP substrate is used after immuno blotting of the prosol. Rinse the membrane and TBST for 15 minutes and twice for five minutes at room temperature. Then re probe the membranes with the primary antibody for the tag to pro.
Im followed by the appropriate secondary antibody. Visualize PRO I and pro Im NNC by detecting chemiluminescence to validate the identity of the bands obtained in the protein membrane overlay assay. The interaction between the proteins A and K and YFP and MP CYFP was assessed in tobacco epidermal cells using the method described in this video as shown here.
When biomolecular fluorescence complementation was assessed strong YFP signal was reconstructed. This BFC signal accumulated in Punta at the cell periphery, which are diagnostic of plasma de because MP is a highly insoluble protein. When expressed in bacteria or in plants, the protein membrane overlay assay was adopted to validate this interaction.
In vitro protein extracts containing one microgram of GST MP or unfused GST were resolved by SDS poly acrylamide gel electrophoresis followed by electro transfer to a nitrocellulose membrane. When these pro iams were probed with soluble a nnk strep two GST mp, but not unfused, GST exhibited binding. Moreover, when the same set of pro IMS were probed with an unrelated pro saw nc IE Arabidopsis Cytoplasmic, NADH kinase tagged strep two, no binding was observed further demonstrating the specificity of the A NK MP interaction While attempting this procedure, it's important to remember to refill the immobilized proteins properly following the presented protocol.
