Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

All experiments were performed in compliance with institutional guidelines. Swine kidney was obtained fresh from the local supermarket. Liver tissues were harvested from C57BL6 wild-type mice maintained at the Institute for Biomedical Aging Research at Innsbruck University, Rennweg 10, 6020 Innsbruck, Austria under the supervision of Univ.-Doz. Dr. Pidder Jansen-Dürr, covered by ethical permission as project leader issued in 2013 (BMWF-66.008/0007-II/3b/2013). Maintenance and use of the mice for the project are covered under ethical permission No. 2020-0.242.978 from May 5th, 2020, issued by the Austrian Ministry for Education, Science, and Research (BMBWF).

1. Preparations

NOTE: Before the protocol starts, several things need to be prepared, i.e., the protein lysis buffer, the crude tissue sample, and a specific antibody, besides general chemicals, and materials.

1. Prepare 250 mL of protein lysis buffer per 100 g of net weight of tissue: 250 mL of 1x PBS with 50 mM NaF, 1 mM PMSF, 2 µg/mL aprotinin, and 1 mM activated orthovanadate (see Table 1). Filter the solution using a 0.22 µm syringe filter unit.
   NOTE: Activation of orthovanadate is required before use to convert it into a more potent inhibitor of protein tyrosine phosphatases¹⁸. Activated orthovanadate may be obtained from commercial suppliers, but also prepared as follows.
   1. Prepare a 200 mM stock solution of (sodium) orthovanadate in ddH₂O. For preparing 10 mL of solution, add 368 mg of Na₃VO₄ to 9 mL of water and dissolve by stirring. Once dissolved, make up the volume to 10 mL with ddH₂O.
      NOTE: The starting pH of the sodium orthovanadate solution may vary with the source of material, and the pH needs to be adjusted to 10 in a repetitive approach as follows.
   2. Depending on the initial pH of the solution, adjust the pH to 10 with NaOH or HCl. At pH > 10, the solution will have a yellow color. Boil the solution until it turns colorless, cool it down to room temperature, and check the pH. If pH is >10, add a small volume of HCl to adjust the pH to 10. At this point, the solution may turn yellow again.
   3. Repeat the boiling and cooling until the solution remains colorless and the pH stabilizes at 10 (approximately 5-7 times). At this point, adding HCl results in a faint appearance of yellow color in the solution. Store activated orthovanadate in 1 mL aliquots at -20 °C.
2. Prepare tubes with 2 mL of lysis buffer per gram of tissue and place them on ice.
   NOTE: This protocol used eight 50 mL tubes, each filled with 30 mL of lysis buffer in total for one swine kidney (about 100-150 g), and two tubes each filled with 40 mL of lysis buffer for 20 mouse livers (each 1-2 g) in total.
3. Prepare the tissue: dissect the tissue on a pre-cleaned glass plate placed on ice in a polystyrene foam box. Cut tissue pieces of about 100 mg each to be easily transferred into respective tubes for subsequent lysis. Transfer the tissue pieces into the prepared tubes (step 1.2).
4. Prepare a saturated ammonium sulfate solution: heat 500 mL of ddH₂O to 70 °C and while stirring, gradually add ammonium sulfate powder (see Table of Materials) until no more ammonium sulfate is dissolved. Cool this (over)saturated solution to room temperature and store it at 4 °C overnight.

2. Total protein extraction

NOTE After preparing the sample in cold protein lysis buffer (see step 1.3), homogenize the tissue as best as possible via sonication by an ultrasonic probe, or using an electric homogenizer as follows.

1. Homogenization of tissues
   1. In the case of a swine kidney, sonicate the suspension preferably by an ultrasonic probe while keeping the sample on ice (10 cycles of 15 s pulse, with intervals of 30 s between the pulses to cool the sample on ice, at medium amplitude with 50% duty cycle).
   2. In the case of mouse organs, homogenize the suspension using an electric homogenizer (starting with a low force, and slowly accelerating to medium force) while keeping the sample on ice. Regularly wash the electric homogenizer in PBS to remove any organic material clogging the device.
   3. Take 20 µL out of the samples and check under the microscope whether the cells of the homogenized tissue are properly destroyed; otherwise, repeat the homogenization.
2. Centrifuge the tubes in a tabletop centrifuge at 10,000 x g for 30 min at 4 °C.
   NOTE: Optionally, centrifuge the supernatant a second time at 20,000 x g for 30 min at 4 °C to eliminate small fractions of the initial pellet that may have been transferred. This will simplify the subsequent filtration in step 2.3.
3. Collect the supernatant in a fresh tube and place it on ice. Sequentially filter the supernatant using 0.45 µm and 0.22 µm syringe filter units. Aliquot the supernatant into 10 mL batches and freeze them at -20 °C for short-term storage or at -80 °C for longer storage.
   NOTE: Pre-filtering with 0.45 µm removes the majority of the particles before a second filtering step with 0.22 µm removes the finer particles. Using the 0.22 µm filter directly can cause the risk of clogging the filters.
4. Prepare a 50 µL sample for SDS-PAGE/western blot analysis by adding 10 µL of 5x SDS sample buffer (see Table 1) to 40 µL of the supernatant, and then boiling at 95 °C for 10 min.
   1. Optionally, resuspend about 100 µL of pellet obtained in step 2.2 in 900 µL of ddH₂O, and prepare a sample for SDS-PAGE/western blot analysis as described above.
      NOTE: Inclusion of pellet-derived samples in the western blot analysis, in addition to the positive control, will indicate whether the expression of the protein is low, or the antibody is problematic.

3. SDS-PAGE and western blot analysis

NOTE: Western blot analysis is required to check for protein solubility. The following describes a protocol for electroblotting, using a Wet/Tank blotting system (see Table of Materials). An alternate protocol for SDS-PAGE may be found elsewhere¹⁹.

1. Prepare a discontinuous 12.5% polyacrylamide SDS-PAGE gel according to the manufacturer's instructions (i.e., a stacking gel on top of a resolving gel; see Table 1). Run the samples previously prepared during step 2 (similar to steps 4, 5, and 6; see below).
   1. Load a protein marker ladder into the first well (see Table of Materials). Load 5 ng of hFAHD1 recombinant protein (obtained from bacteria¹²; see Table 1) as a positive control into the second well.
   2. Subsequently, load 20 µL of the sample to be analyzed, and fill all the remaining wells with 20 µL of prepared SDS-PAGE 1x sample buffer (i.e., 5x sample buffer diluted with ddH₂O). Run the SDS-PAGE gels at 125 V using the SDS-running buffer (see Table 1).
2. After SDS-PAGE is complete, perform a western blot analysis and probe the membranes using the available antibody raised against FAHD1 (see Table 1).
   NOTE: As the samples are taken from crude tissue homogenate, usually the quality of the SDS-PAGE and the western blot analysis at this point is compromised; however, it is important to check whether the protein to extract is soluble in the supernatant. The following protocol was tested for swine kidney, and different mouse organs, including liver, heart, brain, and kidney.
   1. Prepare the 10x western blot transfer buffer (see Table 1). Prepare the 1x western blot transfer buffer (see Table 1) and cool it to 4 °C.
   2. Activate a PVDF membrane for 2 min in methanol. Wash the membrane in ddH₂O for 2 min. Equilibrate the membrane for 15 min in 1x western blot transfer buffer.
   3. Wash the SDS-gel with 1x PBS for 10 min while shaking to remove the SDS-running buffer, and then incubate the gel in 1x western blot transfer buffer for 10 min for equilibration. Assemble the electroblotting cassette (i.e., combining the activated PVDF membrane and gels) according to the manufacturer's instructions.
   4. Run the blot via electroblotting at 300 mA for 1 h in a polystyrene foam box filled with ice or in the cold room (4 °C). Transfer the PVDF membrane into a 50 mL tube with its exposed side facing the inner side of the tube. Incubate the membrane in 20 mL of western blot blocking buffer (see Table 1) overnight at 4 °C while rolling on a tube roller (see Table of Materials).
   5. The next day, wash the membrane for 5 min with 20 mL of western blot washing buffer (PBS with 0.1% (v/v) Tween 20) in the same tube while rolling. Incubate the membrane in the same tube with the primary antibody² (targeting FAHD1; see Table 1) diluted 1:500 in western blot blocking buffer for 1 h at room temperature while rolling.
   6. Wash the membrane in the same tube three times for 10 min each with 20 mL of western blot washing buffer while rolling. Incubate the membrane for 30 min at room temperature with HRP-conjugated secondary antibody (see Table of Materials) diluted 1:3000 in 5 mL of western blot blocking buffer.
   7. Wash the membrane in the same tube three times for 10 min each with 20 mL of western blot washing buffer and twice for 5 min each with 1x PBS. Dry the membrane by carefully holding it with tweezers on one edge, and by touching a piece of cellulose or a piece of Whatman paper with the opposite (lower) edge of the membrane. Put the membrane (exposed side up) on a cleaned glass plate.
   8. Carefully cover the whole membrane with 1 mL of prepared ECL western blot substrate using a pipette, taking care not to create any air bubbles. Let the ECL solution incubate for 3 min, and immediately develop the membrane using X-ray film or using an imaging system.
      NOTE: If the protein was detected in none of the samples but only in the positive control, this may indicate that the protein is insoluble, or not present in adequate amounts to be detected by the antibody. If only nanograms of the positive control were loaded, the first scenario is more likely. If no protein was detected at all, check the quality of the antibody, and maybe switch to a polyclonal antibody rather than a monoclonal antibody. In rare occasions, i.e., for some hydrophobic proteins, the protein may be detectable after centrifugation, but not after filtration. In such a case, it is recommended to use special filter units for hydrophobic proteins.
3. Optionally, stain the PVDF membranes after western blot to control the successful transfer of the protein from the SDS-PAGE gel to the PVDF membrane.
   NOTE: Coomassie staining is recommended for troubleshooting, method development, and documentation, but mind that after applying this protocol, membranes are lost to further western blot analysis. Ponceau S staining gives weaker staining but may be used if the membranes are to be re-probed.
   1. Prepare small trays containing the staining (Coomassie or Ponceau S) and de-staining solutions.
   2. Using tweezers, put the membrane into the staining solution and gently shake until the membrane is stained well (5-10 min).
   3. Transfer the membrane into the destaining solution and shake until the solution is saturated (5-10 min). Repeat the destaining step until the protein bands can be observed on the membrane; if no bands are observed at all, repeat the staining with a longer incubation time. Dry the membrane by placing it on a glass plate using tweezers.

4. Testing: Ammonium sulfate precipitation

NOTE: Ammonium sulfate precipitation is a method of protein purification by altering the solubility of the protein. In a preliminary experiment, the ammonium sulfate concentration is sequentially increased to a value that precipitates a maximum amount of protein contaminants, while leaving FAHD1 in solution. The solubility of the protein is again probed via western blot analysis.

1. Proceed from step 2.3: either thaw an aliquot of the sample or proceed directly after protein extraction (i.e., without freezing the sample). Filter the sample using a 0.22 µm filter unit to exclude possible precipitates after thawing. Prepare six 1.5 mL tubes on ice, and transfer 250 µL of sample into each tube.
2. Prepare a dilution series of 5%, 10%, 15%, 20%, 25%, and 30% ammonium sulfate in the tubes prepared above, and make up the final volume to 1000 µL with protein lysis buffer. Incubate the samples at 4 °C overnight on a tube rotator (see Table of Materials).
3. Using a tabletop centrifuge, centrifuge at 10,000 x g for 30 min at 4 °C and carefully transfer all of the supernatants into separate tubes. Air-dry the resulting pellets and resuspend each of them in 1000 µL of ddH₂O.
4. For each pair of resuspended pellet and supernatant from the previous step, mix 40 µL with 10 µL of 5x SDS sample buffer and boil at 95 °C with open lids until most of the liquid has vaporized. Then, resuspend the pellet in a mixture of 50% DMSO in ddH₂O.
5. Perform SDS-PAGE (step 3) but run the gels at 80 V for 3 h. For each concentration of ammonium sulfate, load the samples derived from the resuspended pellet and supernatant (step 4.3) in pairs. Perform a western blot analysis (step 3).
6. Check for the highest concentration of ammonium sulfate, at which the protein to be purified (i.e., FAHD1) remains in the sample derived from the supernatant. Based on the results, define an ammonium sulfate precipitation protocol for the protein of interest, to be used in future experiments.
   NOTE: Ammonium sulfate is well known to distort SDS-PAGE and western blot. As the concentration of ammonium sulfate increases, the quality of the western blot analysis will be compromised. However, as with step 3 before, this analysis is used to check for the solubility of the protein of interest at given concentrations of ammonium sulfate. This protocol aims to precipitate other proteins, while the protein to be purified must remain soluble.

5. Testing: ionic exchange chromatography with FPLC

NOTE: Molecules with charged functional groups are bound to a silica particle column for FPLC, enabling the differentiation of proteins according to their surface charge. Perform this step twice, using the cationic exchange column and anionic exchange column (see Table of Materials). The protocol steps are the same for either cationic or anionic exchange chromatography, but the buffers to be used are different (see Table 1); both with "low salt" 15 mM NaCl and "high salt" 1 M NaCl conditions. For the columns used, a flow rate of 1 mL/min is recommended.

1. Set up the FPLC system with the anionic or cationic exchange column. Wash the column with 5 column volumes (CVs) of 20% EtOH (in H₂O), followed by 5 CVs of ddH₂O. Alternately, wash the column with 1 CV of low salt buffer, high salt buffer, and again low salt buffer in the order until no more peaks are observed in the chromatogram, but wash at least once.
2. After determining the optimal protocol for ammonium sulfate precipitation on the small scale (step 4), apply the precipitation protocol to 10 mL of original tissue homogenate (step 2). Optionally, dialyze the sample against the low salt buffer.
   1. Apply the sample onto the column (e.g., by injection or by using a sample pump) and collect the flow-through. Wash the column with 1 CV of the low salt buffer.
3. Set up a linear gradient elution from 100% low salt buffer/ 0% high salt buffer to 0% low salt buffer/100% high salt buffer within 3 CVs. Continuously collect 1 mL fractions. After the gradient has finished, continue to run with the high salt buffer until no more protein-associated peaks (UV absorption at 280/255 nm) are detected in the chromatogram over the range of 1 CV.
4. Apply 1 mL of 25% SDS dissolved in 0.5 M NaOH (in ddH₂O) to clean the column. Consecutively, wash the column with 3 CVs of ddH₂O and 3 CVs of 20% EtOH (in ddH₂O).
5. Collect SDS-PAGE samples of all peak-fractions and the flow-through, and probe them via western blot for the presence of the protein of interest (step 3). Snap-freeze the collected fractions in liquid nitrogen and store them at -80 °C.
6. After western blot analysis is complete, thaw and pool the fractions containing the protein of interest and discard the others. Repeat steps 5.1-5.5 with the alternate column (i.e., cationic or anionic exchange column).
7. After both columns have been probed, define an FPLC protocol for the protein of interest, to be used in future experiments. Reduce the volume of the protein solution using ultra-centrifugation filter units (10 kDa, see Table of Materials) down to 2 mL.
   NOTE: There are two expected outcomes of this series of experiments. Either the protein of interest has attached to one of the columns, and the protein solution is already quite pure after elution, or the protein remained in the flow-through in both cases. In the latter scenario, although the protein is in the flow-through, the cleaning effect of this step might still be significant. In such a case, as for FAHD1 in swine kidney and mouse liver, this step of ionic exchange will still be performed. If neither the cationic or anionic exchange column can provide a proper cleaning effect, one may try to modify the pH of the lysate and buffer, and to dialyze the sample against the running buffer before application to FPLC.

6. Protein extraction using defined sub-protocols for ammonium sulfate-precipitation and FPLC

NOTE: Porous particles in a silica gel column for FPLC (See Table of Materials) enable the differentiation of proteins according to their hydrodynamic radius. The described steps are to be performed with an FPLC system, using size exclusion chromatography (SEC). For the SEC column used (see Table of Materials), a flow rate of 0.3 mL/min is recommended.

1. Prepare all the required materials (see step 1), and extract the total protein from the tissue (see step 2). Perform an ammonium sulfate precipitation with all the tissue homogenate that was not used for testing (see step 4). For larger volumes, concentrate the lysate using ultra-centrifugation filter units (10 kDa; see Table of Materials) down to a smaller volume of 50 mL or less.
2. Perform a first purification step using ionic exchange chromatography (see step 5).
   1. Prepare samples for western blot, as described in the previous steps. Perform western blot analysis and pool all FAHD1 containing fractions from ionic exchange chromatography.
   2. Reduce the volume of the protein solution down to 2 mL using ultra-centrifugation filter units (10 kDa). Sequentially filter the solution with 0.45 µm and 0.22 µm syringe filter units to remove any micro-precipitation.
3. Equilibrate the SEC column with 1 CV of SEC running buffer (see Table 1), containing 1 mM DTT. Load the sample onto the column and run the chromatography until all the proteins are eluted (1-2 CV).
   1. Collect fractions of 1 mL of the flow-through that corresponds to significant peaks in the chromatogram (UV absorption at 280/255 nm) and prepare 50 µL samples of each collected fraction for SDS-PAGE and western blot analysis, as described in the previous steps. Snap-freeze all the fractions using liquid nitrogen, and store them at -80 °C.
4. Consecutively wash the SEC column with 1 CV of ddH₂O and 1 CV of 20% EtOH (in ddH₂O). Perform western-Blot analysis, and pool all FAHD1 containing fractions. Reduce the volume of the protein solution down to 2 mL using ultra-centrifugation filter units (10 kDa, see Table of Materials).
5. Assess the protein concentration using a commercial BCA assay kit (see Table of Materials).
   NOTE: The pH and salt content of the mobile phase may influence the elution profile of globular proteins²⁰. Acidic or basic conditions may result in peaks being less defined and increased protein-matrix interactions leading to partial retention of protein on the column²⁰. This effect may be exploited for further protein purification. A repetition of step 6 with different flow rates, pH, and salt concentrations may enhance the purity of the protein²⁰.

7. Silver staining

NOTE: Silver staining analysis of SDS-PAGE gels is required to check for protein contaminations that may not be seen with Coomassie staining. The following protocol is one among many versions that can be found in the literature²¹. Perform all the incubation steps by shaking in a clean glass tray. Collect all silver- and formaldehyde-containing liquids in a special waste container and properly discard them.

1. Incubate the SDS-PAGE gels in silver staining fixing solution (see Table 1) overnight in the cold room. Incubate the gels in silver staining incubation solution (see Table 1) for 3 h at room temperature. Optionally, add glutaraldehyde (see Table 1) to improve the detection of faint bands. Wash the gels four times in ddH₂O for 10 min each.
2. Incubate the gels in silver staining silver solution (see Table 1) for 1 h.
   NOTE: Mind that from now on all the liquids and the gel itself contain silver and formaldehyde which are toxic.
3. Incubate the gels in silver staining developer solution (see Table 1) with vigorous shaking until bands are clearly visible. To stop the reaction, discard the developer solution and immediately incubate the gels in silver staining stop solution (see Table 1) for a minimum of 10 min.
   NOTE: Bands stained in steps 7.2 and 7.3 will constantly become more developed. Adding more formaldehyde to the solution than stated may be necessary if the staining is weak.