Team:Goettingen/protocol protein

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8. Run the gel at 80 V for 10 minutes (sample will pass the stacking gel) and then turn up to 120 V.<br />
8. Run the gel at 80 V for 10 minutes (sample will pass the stacking gel) and then turn up to 120 V.<br />
9. Pry the plates apart with a gel spacer. Gels can be stained with Coomassie (or Silver Stain) to visualize the proteins.<br />
9. Pry the plates apart with a gel spacer. Gels can be stained with Coomassie (or Silver Stain) to visualize the proteins.<br />
-
 
             </p>
             </p>
-
          
+
<h3 id="Coo">Coomassie Staining</h3>
 +
<p>
 +
1. Place the gel in plastic container and incubate in fixation solution for 10 min with agitation. <br />
 +
2. Remove fixation solution and cover the gel with staining solution, shake for 10 - 20 min at RT.<br />
 +
3. Remove staining solution (re-usable). Wash gel with destaining solution over night. Boiling the gel in a microwave and addition of a paper towel will increase destaining. <br /><br />
 +
</p>
 +
<h3 id="P_Exp">Test expression of recombinant proteins in<i> E. coli</i> BL21(DE3)</h3>
 +
<p>
 +
 
 +
1. Transform the desired plasmid into the <i>E. coli</i> strain BL21blue and plate on LB + Ampicillin plates; transform the same <i>E. coli</i> with empty vector as negative control.<br />
 +
2. Pick a grown colony from the transformation plate and inoculate 10 ml LB medium.<br />
 +
3. Grow the culture at 37°C until an OD600 ~ 0.8 and induce protein expression with 1 mM IPTG.<br />
 +
4. Incubate for additional 3 hours at 37°C. <br />
 +
5. Harvest small aliquots before induction and every hour after induction. Collect a volume of cells according the following formula: <br />
 +
µl sample=  (100 [µl])/OD600<br />
 +
E.g. If you measure an OD600 of 1.87, you have to take 53.3 µl of the cell culture.<br />
 +
6. Spin down cells for 5 min at 13000 rpm. Discard supernatant. Store the cells at -20 °C until all samples are collected.<br />
 +
7. Boil the samples in 15 µl SDS loading dye for 15 min at 95 °C. <br />
 +
8. Run SDS-PAGE.<br /><br />
 +
 
 +
</p>
 +
<h3 id="P_Strep">Expression of Strep-tagged proteins </h3>
 +
<p>
 +
1. Inoculate 100 ml LB medium with the desired strains and grow at 37°C or lower temperatures over night.<br />
 +
2. Next day, inoculate 1 L LB medium to an OD600 of 0.2. Incubate the cells for 60 min to an OD600 = 0.8 at conditions suitable for the protein of interest.<br />
 +
3. Add IPTG to a final concentration of 1 mM to induce recombinant protein expression. <br />
 +
4. Wait 3 - 4 hours for the protein expression. Then centrifuge 500 ml of each sample in a suitable centrifugation tube at 5000 rpm and 4°C for 15 min. Discard supernatant.<br />
 +
5. Pellets can be stored at -20°C till next day. Resuspend the pellet in 10 ml cold buffer W. <br />
 +
6. Now, cells can be used for lysis by French press or sonification.<br /><br />
 +
 
 +
</p>
 +
<h3 id="C_dis">Cell disruption by French Press & separation of cell debris</h3>
 +
<p>
 +
1. Resuspend a cell pellet from 500 ml culture in 10 ml cold Buffer W. Mix by pipetting or vortexing.<br />
 +
2. Adjust a pressure of 18.000 psi for <i>E. coli</i>.<br />
 +
3. Fill cells in and close the press tightly. After disruption, let the cells flow out slowly and cool the falcon on ice.<br />
 +
4. For separation of proteins and cell debris, fill the disrupted cells into tubes for ultracentrifugation. <br />
 +
5. Weight each tube to balance them exactly. <br />
 +
6. Centrifuge at 35000 rpm and 4°C for 60 min. <br />
 +
7. Collect the supernatant and discard pellet. <br /><br />
 +
 
 +
</p>
 +
<h3 id="Puri">Purification with Strep-Tactin ® resins</h3>
 +
<p>
 +
1. Pack columns with 1 ml 50 % Strep-Tactin solution - here: 1 column bed volume (CV) = 0.5 ml.<br />
 +
2. Equilibrate the column with 2 CV Buffer W.<br />
 +
3. Apply the protein extract to the columns. Collect the flow through (FT).<br />
 +
4. Wash column 4 times with 1 CV of Buffer W. Collect the wash fractions for SDS-gel.<br />
 +
5. Elute the protein 4 times with 0.5 CV of Buffer E. Collect elution fractions for SDS-gel.<br />
 +
6. Regenerate the columns by washing 3 times with 5 CV of Buffer R. The color will change from yellow to red.<br />
 +
7. Remove HABA and equilibrate by adding 2 times 4 CV of Buffer W. Overlay the columns with 2 ml Buffer W and store at 4 °C.<br /><br />
 +
 
 +
</p>
 +
<h3 id="Dialysis">Control & Dialysis of protein samples</h3>
 +
<p>
 +
1. Thaw/Store all collected protein samples on ice. <br />
 +
2. Determine the concentration of proteins in crude extract (CE) and flow trough (FT) with a Bradford Assay and use a total amount of 5 µg protein for SDS-PAGE. Use 15 µl from all other collected samples. Run SDS-PAGE.<br />
 +
3. Pool the fractions of proteins that contain the largest protein amount and dialyze them.<br />
 +
4. Cut dialyze tube in 8 cm long stripes, boil them in water for 10 minutes. Then, wash the tubes in water to get rid of heavy metals (wear gloves).<br />
 +
5. Close one side of the dialyze tubes with a zipper, fill in the pooled protein fractions and close the other site as well.<br />
 +
6. Label your packages well! (You can use a labeled 1.5 ml E-cup and clip it to one corner).<br />
 +
7. Put the packages into 1000 CV of your desired buffer and incubate at 4 °C over night. This step can be repeated. Additionally, you can add DTTup to 100 mM to avoid the oxidation of your protein.<br />
 +
8. Pipette carefully the proteins out of the packages and measure the protein concentration with a Bradford Assay. <br /><br />
 +
 
 +
</p>
 +
<h3 id="Bradford">Bradford Assay</h3>
 +
<p>
 +
1. Use RotiQuant (= Bradford) solution and dilute it 1:5 with H2O.<br />
 +
2. Use 1 ml Bradford solution in a 1.5 ml reaction tube (per protein sample) and take different volumes of your protein (e.g. 0.5, 1, 2 µl) to determine the concentration.<br />
 +
3. Prepare the same volumes of buffer in Bradford solution as reference.<br />
 +
4. Vortex the tubes and incubate for 5 minutes at room temperature.<br />
 +
5. Measure the OD at 595 nm. Do not forget to blank! <br />
 +
6. Calculate the protein concentration with the given formula:<br />
 +
A_595nm= a (ml/mg)x c (mg/ml); a=0.0536<br /><br />
 +
 
 +
</p>
 +
         <br /><br />
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Revision as of 21:29, 15 October 2014

SDS-PAGE

1. Clean the glass plates, combs and mats with EtOH and assemble the glass plates.
2. Wear gloves while making the gels (Acrylamid is neurotoxic). Mix the ingredients of the running gel and pour the mixture between the assembled glass plates. Overlay the running gel with Ethanol or Isopropanol to make sure that surface is horizontal.
3. After polymerisation, remove the alcohol and dry with Whatman-paper. Mix the ingredients and pour it on top of the running gel. Insert a comb immediately.
4. After polymerisation, clamp the gel into the electrophoresis chamber and fill the chamber with 1x running buffer. Remove the comb and wash the wells with running buffer using a pipette.
5. The protein samples (up to 15 µl) are mixed with SDS-loading dye (2.5 µl) and incubated at 95°C for 10 minutes.
6. Briefly centrifuge the samples.
7. Load the samples with a pipette into the wells of the gel. Do not forget the MARKER.
8. Run the gel at 80 V for 10 minutes (sample will pass the stacking gel) and then turn up to 120 V.
9. Pry the plates apart with a gel spacer. Gels can be stained with Coomassie (or Silver Stain) to visualize the proteins.

Coomassie Staining

1. Place the gel in plastic container and incubate in fixation solution for 10 min with agitation.
2. Remove fixation solution and cover the gel with staining solution, shake for 10 - 20 min at RT.
3. Remove staining solution (re-usable). Wash gel with destaining solution over night. Boiling the gel in a microwave and addition of a paper towel will increase destaining.

Test expression of recombinant proteins in E. coli BL21(DE3)

1. Transform the desired plasmid into the E. coli strain BL21blue and plate on LB + Ampicillin plates; transform the same E. coli with empty vector as negative control.
2. Pick a grown colony from the transformation plate and inoculate 10 ml LB medium.
3. Grow the culture at 37°C until an OD600 ~ 0.8 and induce protein expression with 1 mM IPTG.
4. Incubate for additional 3 hours at 37°C.
5. Harvest small aliquots before induction and every hour after induction. Collect a volume of cells according the following formula:
µl sample= (100 [µl])/OD600
E.g. If you measure an OD600 of 1.87, you have to take 53.3 µl of the cell culture.
6. Spin down cells for 5 min at 13000 rpm. Discard supernatant. Store the cells at -20 °C until all samples are collected.
7. Boil the samples in 15 µl SDS loading dye for 15 min at 95 °C.
8. Run SDS-PAGE.

Expression of Strep-tagged proteins

1. Inoculate 100 ml LB medium with the desired strains and grow at 37°C or lower temperatures over night.
2. Next day, inoculate 1 L LB medium to an OD600 of 0.2. Incubate the cells for 60 min to an OD600 = 0.8 at conditions suitable for the protein of interest.
3. Add IPTG to a final concentration of 1 mM to induce recombinant protein expression.
4. Wait 3 - 4 hours for the protein expression. Then centrifuge 500 ml of each sample in a suitable centrifugation tube at 5000 rpm and 4°C for 15 min. Discard supernatant.
5. Pellets can be stored at -20°C till next day. Resuspend the pellet in 10 ml cold buffer W.
6. Now, cells can be used for lysis by French press or sonification.

Cell disruption by French Press & separation of cell debris

1. Resuspend a cell pellet from 500 ml culture in 10 ml cold Buffer W. Mix by pipetting or vortexing.
2. Adjust a pressure of 18.000 psi for E. coli.
3. Fill cells in and close the press tightly. After disruption, let the cells flow out slowly and cool the falcon on ice.
4. For separation of proteins and cell debris, fill the disrupted cells into tubes for ultracentrifugation.
5. Weight each tube to balance them exactly.
6. Centrifuge at 35000 rpm and 4°C for 60 min.
7. Collect the supernatant and discard pellet.

Purification with Strep-Tactin ® resins

1. Pack columns with 1 ml 50 % Strep-Tactin solution - here: 1 column bed volume (CV) = 0.5 ml.
2. Equilibrate the column with 2 CV Buffer W.
3. Apply the protein extract to the columns. Collect the flow through (FT).
4. Wash column 4 times with 1 CV of Buffer W. Collect the wash fractions for SDS-gel.
5. Elute the protein 4 times with 0.5 CV of Buffer E. Collect elution fractions for SDS-gel.
6. Regenerate the columns by washing 3 times with 5 CV of Buffer R. The color will change from yellow to red.
7. Remove HABA and equilibrate by adding 2 times 4 CV of Buffer W. Overlay the columns with 2 ml Buffer W and store at 4 °C.

Control & Dialysis of protein samples

1. Thaw/Store all collected protein samples on ice.
2. Determine the concentration of proteins in crude extract (CE) and flow trough (FT) with a Bradford Assay and use a total amount of 5 µg protein for SDS-PAGE. Use 15 µl from all other collected samples. Run SDS-PAGE.
3. Pool the fractions of proteins that contain the largest protein amount and dialyze them.
4. Cut dialyze tube in 8 cm long stripes, boil them in water for 10 minutes. Then, wash the tubes in water to get rid of heavy metals (wear gloves).
5. Close one side of the dialyze tubes with a zipper, fill in the pooled protein fractions and close the other site as well.
6. Label your packages well! (You can use a labeled 1.5 ml E-cup and clip it to one corner).
7. Put the packages into 1000 CV of your desired buffer and incubate at 4 °C over night. This step can be repeated. Additionally, you can add DTTup to 100 mM to avoid the oxidation of your protein.
8. Pipette carefully the proteins out of the packages and measure the protein concentration with a Bradford Assay.

Bradford Assay

1. Use RotiQuant (= Bradford) solution and dilute it 1:5 with H2O.
2. Use 1 ml Bradford solution in a 1.5 ml reaction tube (per protein sample) and take different volumes of your protein (e.g. 0.5, 1, 2 µl) to determine the concentration.
3. Prepare the same volumes of buffer in Bradford solution as reference.
4. Vortex the tubes and incubate for 5 minutes at room temperature.
5. Measure the OD at 595 nm. Do not forget to blank!
6. Calculate the protein concentration with the given formula:
A_595nm= a (ml/mg)x c (mg/ml); a=0.0536