The overall goal of this procedure is to covalently bind BMP two molecules to surfaces without losing their bioactivity. This is accomplished by first forming a gold layer on a substrate by physical vapor deposition. The second step is to create a self-assembled monolayer of mu NHSA bifunctional NHS Thiol linker.
Next, RH BMP two is covalently bound to the linker. The final step is to remove the non bound BMP two by sonication. Ultimately, an antibody binding assay is used to show the successful binding of BMP two onto the surfaces and the biological activity of surface bound BMP two is determined in cells by analyzing SMA 1 5 8 phosphorylation.
This method can help answer key questions in the field of growth factor biology, such as if the initial binding of growth factors to their receptors at the cell surface is sufficient to trigger signaling responses. Demonstrating the procedure will be two members of my laboratory, Teresa Paul, and Elizabeth Schwab. To begin this procedure, clean glass cover slips with precision wipes and sonicate them for five minutes in a one-to-one mixture of ethyl acetate and methanol.
After sonication, rinse the substrates with methanol and dry them under nitrogen flow. Place the clean substrates in a sputter coating device and evacuate the chamber. Remove the uppermost chromium oxide layer from the chromium target by sputtering for 60 seconds.
Then coat the substrate with a 10 nanometer chromium layer followed by coating with a 40 nanometer gold layer. When finished, remove the gold substrates from the chamber and set them aside. Transfer the gold substrates into a two neck round bottom flask and incubate them in one millimolar M-U-N-H-S in DMF at room temperature for four hours under nitrogen atmosphere.
After incubation, sonicate the substrates in DMF for two minutes. Rinse them with DMF and methanol and dry them under nitrogen flow. Dilute the RHB MP two stock solution with one molar sodium chloride in PBS to give a final concentration of 3.5 micrograms per milliliter.
Adjust this working solution to pH 8.5 with sterile 10 millimolar potassium hydroxide immediately prior to use. Place A-P-D-M-S ring on top of the sample. Transfer the substrate to a six well plate to ensure that only the substrate surfaces are exposed to the incubation solution.
Then incubate the functionalized M-U-N-H-S substrates with the RHB MP two working solution at four degrees Celsius overnight. After removing the incubation supernatant, add one molar sodium chloride in PBS and sonicate the substrates for two minutes. When finished, wash the substrates three times with sterile one molar sodium chloride in PBS to block unspecific absorption of antibody.
Incubate the substrates in 5%BSA in PBS for one hour at room temperature. Then incubate the substrates with an anti BMP two antibody solution for one hour at room temperature when finished, wash the substrates twice with PBS and sonicate them for 30 seconds. Next, incubate the substrates in an HRP conjugated secondary antibody solution for 30 minutes at room temperature and then wash them twice with PBS.
Use the amply flu red assay to measure HRP enzymatic activity at 570 nanometers with a plate reader seed one times 10 to the fifth mouth C two C 12 myoblasts per well in six well plates in growth medium and incubate them for 24 hours at 37 degrees Celsius and 5%CO2. Once the plates have been removed from the incubator, aspirate the media from the wells and starve the cells in serum free DMEM for three to five hours prior to seeding onto the functionalized BMP two substrate surfaces for the investigation of short-term signaling induction. Replace the medium with 200 microliters of fresh serum free DMEM and place the immobilized BMP two substrates above the cells using fine tip tweezers following incubation at 37 degrees Celsius and 5%CO2.
Remove the substrates gently and aspirate the medium with a pipette. Then wash the cells twice with PBS and proceed to the analysis of cell responses using gel electrophoresis and western blotting for the analysis of long-term biological activity. Plate the cells onto the surfaces as previously described, and culture them at 37 degrees Celsius and 5%CO2 in 2%FBS for six days.
After incubation. Image the cells by fluorescence microscopy to verify RH BMP two binding surfaces presenting immobilized RH BMP two were incubated with a BMP two specific antibody and a secondary antibody conjugated with HRP. The surface immobilized RH BMP two or I BMP two was detected before and after cell stimulation.
Shown here is the successful binding of RH BMP two to the surface in a confirmation recognized by the anti BMP two IgG. The surface immobilized RH BMP two was quantified using the amli flu red chole metric assay and the data shows that after cell stimulation, the protein was still detected on the surface cell responses to surfaces presenting immobilized RHB MP two were evaluated and compared to the effect of non-treated gold substrates. Here, C two C 12 cells were stimulated for 30 minutes by RHB MP two functionalized surfaces approaching from the top.
In this model, BMP two inhibits the differentiation of cells into multinucleated myo tubes and induces osteoblast phenotypes. The activation of SMAD 1 5 8, which are downstream reporters of the BMP two signaling pathway is analyzed by western blotting as shown here. SM phosphorylation is observed after 30 minute stimulation by I BMP two, while no phosphorylation of SMAD 1 58 occurs in cells exposed to untreated gold samples.
This result indicates that the immobilization process does not alter BMP two short-term activity and triggers early steps in smad signaling to determine whether I BM P two affects long-term osteogenic differentiation. The expression level of alkaline phosphatase was investigated by a chole metric assay. Alkaline phosphatase activity of the C two C 12 cells was measured after incubating the cells for six days on plain gold and on IBP two surfaces.
Alkaline phosphatase activity in lysates of cells cultured on I BM P two surfaces showed a significantly higher absorption in comparison to the control. This result reveals that IBMP two induces the expression of alkaline phosphatase in C two C 12 cells to investigate the effect of IBP two. On Myogenesis C two C 12 cells were plated directly on gold or on functionalized surfaces and cultured under low serum conditions.
After six days, staining of myosin heavy chain was performed as shown here. C two C 12 cells failed to form myosin heavy chain positive myo tubes in the presence of I BM P two in contrast to the cells on gold substrates. Following this procedure, other methods like immobilizing BMP two on gold nanoparticles can be performed to answer additional questions.
For example, how much of immobilized BMP two is locally needed to efficiently trigger signaling responses.
