2 Refer back to your Isolation and Purification of GFP lab. Describe what proteins should be present in each of your gel
Posted: Fri Mar 04, 2022 10:35 am
2 Refer back to your Isolation and Purification of GFP lab. Describe what proteins should be present in each of your gel samples (crude, flow through, wash and elution samples).
What is in the crude, flow through, wash, and elution? protein, pglo, etc.
Protein Chromatography 1. Prepare the chromatography column. Remove the caps from the top and bottom of the prefilled HIC column and clamp it to your ring stand. Place an empty test tube or small beaker below the column to collect the storage buffer. 2. Allow the entire liquid (storage) buffer to drain through the column (approximately 3–5 minutes). If the column is not flowing well, ask your instructor for help. Let the liquid flow until there is a very little amount of liquid resting on the top of the resin. Try not to let the resin go dry. 3. Equilibrate the column resin by adding 1 mL of equilibration buffer to the top of the column twice. This is done by adding two 1 mL aliquots separately. Collect and discard this buffer. 4. When the last of the equilibration buffer has reached the surface of the HIC matrix/resin, stop the column by replacing the cap at the bottom until you are ready to load your sample. (If preparing your column on day 1, cap the top as well, label, and give to your instructor.) 5. Label four microtubes 1-4 (or FT, W, E1, E2) along with your name and place the tubes in a rack supplied in your laboratory. When ready to load your sample on the column, place microtube 1 (FT) under your column and proceed. 6. Carefully and gently load all 500 ul of sample (the supernatant + binding buffer) onto the top of the column. Hold the pipette tip against the side of the column wall, just above the upper surface of the matrix, and let the sample drip down the side of the column wall. Collect all of the flow through (FT). Once sample is bound to the column and stops dripping, carefully
examine the column using a UV light (keep the column upright at all times!). Note your observations. If you do not see a glow, check with your instructor about adding more of the other supernatant. After the column stops dripping, transfer the column to microtube 2 (W). 7. Add 250 ul of wash buffer and let the entire volume flow into the column, collecting the wash (W) in microtube 2. Examine the column using the UV light. Note your observations. After the column stops dripping, transfer it to tube 3 (E1). 8. Add 750 ul of TE buffer and let the entire volume flow into the column. Collect the eluent in tube 3 (E1). Examine the column using the UV light. Note your observations. If the GFP is still bound to the column, add an additional 250 ul TE buffer and collect the eluent in tube 4 (E2). 9. Examine all collection microtubes and note any differences in color between the tubes. Label all samples very well and place them in the freezer for further characterization by gel electrophoresis.
- 2 Refer Back To Your Isolation And Purification Of Gfp Lab Describe What Proteins Should Be Present In Each Of Your Gel 1 (426.01 KiB) Viewed 50 times
- 2 Refer Back To Your Isolation And Purification Of Gfp Lab Describe What Proteins Should Be Present In Each Of Your Gel 2 (252.87 KiB) Viewed 50 times
Protein Chromatography 1. Prepare the chromatography column. Remove the caps from the top and bottom of the prefilled HIC column and clamp it to your ring stand. Place an empty test tube or small beaker below the column to collect the storage buffer. 2. Allow the entire liquid (storage) buffer to drain through the column (approximately 3–5 minutes). If the column is not flowing well, ask your instructor for help. Let the liquid flow until there is a very little amount of liquid resting on the top of the resin. Try not to let the resin go dry. 3. Equilibrate the column resin by adding 1 mL of equilibration buffer to the top of the column twice. This is done by adding two 1 mL aliquots separately. Collect and discard this buffer. 4. When the last of the equilibration buffer has reached the surface of the HIC matrix/resin, stop the column by replacing the cap at the bottom until you are ready to load your sample. (If preparing your column on day 1, cap the top as well, label, and give to your instructor.) 5. Label four microtubes 1-4 (or FT, W, E1, E2) along with your name and place the tubes in a rack supplied in your laboratory. When ready to load your sample on the column, place microtube 1 (FT) under your column and proceed. 6. Carefully and gently load all 500 ul of sample (the supernatant + binding buffer) onto the top of the column. Hold the pipette tip against the side of the column wall, just above the upper surface of the matrix, and let the sample drip down the side of the column wall. Collect all of the flow through (FT). Once sample is bound to the column and stops dripping, carefully
examine the column using a UV light (keep the column upright at all times!). Note your observations. If you do not see a glow, check with your instructor about adding more of the other supernatant. After the column stops dripping, transfer the column to microtube 2 (W). 7. Add 250 ul of wash buffer and let the entire volume flow into the column, collecting the wash (W) in microtube 2. Examine the column using the UV light. Note your observations. After the column stops dripping, transfer it to tube 3 (E1). 8. Add 750 ul of TE buffer and let the entire volume flow into the column. Collect the eluent in tube 3 (E1). Examine the column using the UV light. Note your observations. If the GFP is still bound to the column, add an additional 250 ul TE buffer and collect the eluent in tube 4 (E2). 9. Examine all collection microtubes and note any differences in color between the tubes. Label all samples very well and place them in the freezer for further characterization by gel electrophoresis.