The overall goal of this procedure is to efficiently and effectively isolate and characterize chimeric human FC expressing proteins using protein, a membrane absorbers, and a streamlined workflow. This is accomplished by first clarifying the cell culture supernatant with a 0.22 micron filter. The second step is to assemble the flow system and begin perfusion of the cell culture supernatant through the protein A membrane absorber.
Next, the human FC expressing protein is alluded off the membrane absorber with the resulting EIT concentrated through ultra filtration, followed by buffer exchange through dialysis. The final steps are to use flow cytometry and western blotting to determine the quality and quantity of the concentrated protein. Ultimately, protein A membrane absorbers, along with a streamlined workflow show a decrease in processing time and potential increase in protein yield compared to traditional affinity chromatography with protein A aro speed packed columns.
So the main advantage of this technique over existing methods like column affinity chromatography is that the membrane absorber allows for potentially higher yields of the protein with lower processing time. The selection of appropriate characterization methods can also lower processing time, Prepare the membrane absorber following the manufacturer's instructions, ensuring that all solutions perfused through are at room temperature and pre-filtered through a 0.22 micron filter. To begin, fill a 10 milliliter syringe with 0.22 micron filtered KO's phosphate buffered saline or DPBS and discharge any air bubbles.
Ensuring that air never enters the membrane absorber perfuse DPBS to remove the storage solution and to equilibrate the membrane absorber immediately before use. Filter the cell culture supernatant immediately before profusion through the membrane absorber using a vacuum driven 0.22 micron sterile filtration unit to load the protein A membrane absorber, aspirate the cell culture supernatant into a lure lock syringe, and discharge any air bubbles to assemble materials and equipment. First, connect the syringe to the inlet of the membrane absorber.
Attach flexible tubing to the membrane absorber outlet if desired. Place a 0.22 micron syringe filter between the syringe and the membrane absorber. Use a flask or bottle to catch through from the absorber.
Set the syringe pump to the desired volumetric flow rate, but do not exceed the manufacturer's recommended flow rate. Perfu the cell culture supernatant through the membrane absorber collecting flow through. Reload the syringe with supernatant as needed.
If desired, test the flow through for the presence of protein using flow cytometry, western blotting or eisa based on lab preference, it is typically unnecessary to reperfuse the flow through to elute the protein from the membrane absorber. First, wash the membrane absorber with 10 milliliters of DPBS to remove any non bound protein. Elute protein from the membrane absorber at the desired flow rate.
Using 10 to 15 milliliters of elution buffer. Catch the EIT in a tube containing neutralizing buffer at 10%of the elucian volume. After eluding the protein, regenerate the membrane absorber according to the manufacturer's instructions.
Fill the membrane absorber with 20%ethanol in DPBS for long-term storage at four degrees Celsius. Deposit all EIT. In a 10 kilodalton molecular weight cutoff centrifugal filter unit.
Follow the manufacturer's instructions for centrifugation dialyze. The reten Tate. In a small volume dialysis unit with a 10 kilodalton molecular weight cutoff against the buffer of choice buffer may need to be added to the dialyzed material.
If protein precipitation is a concern, resolve purified protein and FC standards on the gel of choice by SDS page under reducing or non reducing conditions as desired. Use a mini gel rather than a mini gel to minimize runtime load multiple sample amounts of purified protein, including samples diluted with DPBS to ensure that the purified protein band signals are within the linear signal range of FC standards. In image analysis after separation on the gel transfer proteins from the gel to a poly olaine fluoride membrane in a discontinuous buffer system using a semi-dry blotter following the manufacturer's instructions, transfer time is typically five to 10 minutes.
Kumasi stain the gel after transfer to verify transfer efficiency. Perform immuno blotting with anti FC or anti IgG conjugated to alkaline phosphatase or horseradish peroxidase using a vacuum assisted protein detection. Immuno blotting time is typically less than one hour.
Develop the immuno blot using the substrate and method of choice. Quantify the purified protein by image analysis. Perform a linear regression on the band signals from FC standards.
If R squared is greater than 0.90, use the equation for the line to calculate the protein quantity from its band signal accounting for sample dilution if necessary. Since quantification of the protein is performed on the basis of fc, adjust the value for molecular weight differences between the protein and fc. If R squared is less than 0.90, repeat the expedited western blotting procedure.
Finally, validate the protein using functional assays or methods to detect FC via flow cytometry. Western blotting or EISA flow cytometry was used to test for the presence of functional GAL one HFC in the starting cell culture supernatant as indicated by the blue histogram fresh cell culture medium, which is shown here in red, served as the negative control overlapping this histogram in green is the flow through indicating that the membrane absorber efficiently captured and retained GAL one HFC gal one HFC was alluded from the membrane absorber in one milliliter increments at one milliliter per minute using an amine based acidic buffer and neutralized elution fractions were subsequently tested for the presence of functional GG one HFC using flow cytometry. GL one HFC signal sequentially increased from elucian one approximately equal to the negative control to a maximum at EEU three, and then decreased to a nearly constant level at ellucians nine and 10.
The final EEU 10 did not reach equivalence to the negative control signal due to some ads absorber retention of GL one HFC quantification and characterization of the purified GL one HFC was performed in a western blotting procedure expedited by semi-dry transfer and vacuum assisted immuno blotting. By comparing GA one HFC signal to HFC quantification standards. Both the quantity and quality of the purified material were determined.
GAL one HFC and degraded GAL one HFC signals were within the standards range. Signal intensities of HFC standard bands were linearly related to the quantity of HFC present. The concentration of purified GAL one HFC on the basis of HFC were determined to be 450 micrograms per milliliter by image processing.
Adjusting for the molecular weight of G one HFC. The concentration of the purified material was 720 micrograms per milliliter. In contrast, too much purified GAL one HFC was loaded in lane six and seven of the western blot shown here, which generated signals that saturated or exceeded the HFC standards range, thereby preventing quantification.
Alternative to western blotting kumasi gel staining can serve as a rapid quality control check and or quantification method. Note that a band at approximately 10 kilodaltons in addition to bands at approximately 25 kilodaltons and 40 kilodaltons were present in the degraded GAL one HFC sample. This band is degraded GAL one HFC that was non-reactive with the detection antibody used in western blots.
After watching this video, you should have a good understanding of how to isolate chimeric human FC expressing proteins using membrane absorbers and how to characterize those proteins in as little as four hours.
