Protein Detection by SDS-PAGE & Western Blot

Transcription

1 Protein Detection by SDSPAGE & Western Blot Buffers Prepare buffers for SDSPAGE and Western Blot. See materials for different buffers below 10x SDS Running Buffer Dissolve 30.0 g of Tris base, 140 g of glycine, and 10.0 g of SDS in 1000 ml of H2O. The ph of the buffer should be 8.3 and no ph adjustment is required. Store the running buffer at room temperature and dilute to 1X before use. 20X TBS Dissolve 48.3 g of Tris base and g of NaCl in 1000 ml. Adjust the ph to 4 using HCl. 1X TBST Mix 50 ml of 20X TBS and 1 ml Tween 20. Fill up to 1L with dh2o. 5% Milk Blocking Buffer Dissolve 5 g of dry milk powder in 150 ml of 1X TBST and mix well. Cell Lysis Lyse cell pellets and extract intracellular components for use in SDSPAGE and Western Blot. Sonicator Cell pellet

2 Protein Detection by SDSPAGE & Western Blot Resuspend the cell pellet in 30 µl distilled water. Sonicate cells for 10 minutes. Protein Deglycosylation Deglycosylate proteins by the cleavage of Nlinked oligosaccharides using the enzyme NGlycosidase F (PNGase F). Protein sample (supernatant or lysed cells) PNGase F glycosidase (Promega) 5% SDS 1M DTT 0.5M sodium phosphate buffer (ph 5) Triton X Add 1 µl of 5% SDS to 20 µl of protein sample. Add 1µl of 1M DTT. Denature sample by heating at 95 C for 5 minutes. Cool sample for 5 minutes at room temperature. Add 2µl of 0.5M sodium phosphate buffer (ph 5). Add 2µl of Triton X100. Add 2µl of recombinant PNGase F. Incubate at 37 C for 1 3 hours. Proceed to visualization of deglycosylation proteins by gelshift on SDSPAGE, with the deglycosylated product running faster than the glycosylated substrate.

3 Protein Detection by SDSPAGE & Western Blot Other buffers than sodium phosphate buffer can be used if they are within the acceptable ph range for PNGase F (ph 6 10). NP40 may be substituted for Triton X100. SDSPAGE Separation of proteins according to size by electrophoresis using a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. Protein sample Any kd MiniPROTEAN TGX Precast Protein Gel (BioRad) PageRuler Prestained Protein Ladder (Thermo Fisher Scientific) 5X sample loading buffer 1X SDS running buffer Add 5X sample loading buffer in a ratio of 1:5 to the protein sample. Incubate samples for 5 minutes in a dry heating block at 95 C. Centrifuge samples at 13,000 rpm for 60 seconds. Remove the tape at the bottom of the precast gel cassette and assemble the cassette into the MiniPROTEAN Tetra cell. Add running buffer to the inner and outer chambers. Remove the comb from the gel. Load the samples (volume depending on well size) and protein ladder (5 µl) into the wells. Run the gel at 120V for 1h or V for 30 to 35 minutes (for the latter, run the gel in a cold room at 4 C), until the dye front reaches the reference line. Remove cassette from the cell and remove the gel from the cassette using opening levers. Carefully wash all equipment used for electrophoresis with water

4 Protein Detection by SDSPAGE & Western Blot Coomassie Blue Staining Visualization of proteins separated by gel electrophoresis using Coomassie Blue dye. SimplyBlue SafeStain (Thermo Fisher Scientific) Gel from electrophoresis Overnight Staining Place the gel in a staining container and add SimplyBlue SafeStain solution until it completely covers the gel. Incubate overnight at room temperature on an orbital shaker. Collect the stain after incubation (it can be reused). Rinse the gel with dh2o. Destain the gel in dh2o on an orbital shaker, changing water multiple times throughout the day until the desired background is achieved. Quick Staining (Microwave Procedure) Place the gel in 100 ml of dh2o in a loosely covered container and microwave on High (950 to 1100 watts) for 1 minute until the solution almost boils. Shake the gel on an orbital shaker for 1 minute. Discard the water. Repeat Steps 1 and 2 two more times. After the last wash, add 20 ml of SimplyBlue SafeStain and microwave on High for 45 seconds to 1 minute until the solution almost boils. Rinse the gel with dh2o. Destain the gel overnight in dh2o on an orbital shaker, until the desired background is achieved. Use caution while using the stain in a microwave oven. Do not overheat the staining solutions. The detection limit is 10 ng BSA. An additional incubation in 20 ml of 20% NaCl can be used to lower the detection limit to 5 ng BSA. The gel can be stored for several weeks in this salt solution.

5 Protein Detection by SDSPAGE & Western Blot Western Blot Visualization of specific proteins separated by gel electrophoresis using antibodies conjugated to HRP. Primary antibody HRPconjugated secondary antibody Gel from electrophoresis TransBlot Turbo Transfer System and Pack, PVDF (BioRad) 5% milk blocking buffer 1X TBST washing buffer After electrophoresis, use the TransBlot Turbo transfer system to transfer the protein bands from the gel to the membrane. Incubate membrane in 2025 ml of blocking buffer for 1 hour at room temperature. Incubate membrane with primary antibody (at an appropriate dilution) for 1 hour at room temperature with gentle agitation or shaking. After completing the incubation wash the membrane 3 times for 5 minutes with 1X TBST. Incubate membrane with HRPconjugated secondary antibody (at an appropriate dilution) for 1 hour at room temperature with gentle agitation or shaking. Again, after completing the incubation wash the membrane 3 times for 5 minutes with 1X TBST. Incubate membrane with chemiluminescent HRP substrate for 30 seconds and detect the protein band with the use of a detection system. Note: do not leave the membrane with substrate for more than 30 seconds. These protocols are based on protocols from igem Stockholm 2017, the MiniPROTEAN TGX Precast Protein Gels Quick Start Guide from BioRad, the SimplyBlue SafeStain manual from Thermo Fisher Scientific, and protocols used by the groups of Johan Rockberg and Qi Zhou at KTH Royal Institute of Technology.