Experiments

Design of GBlock (In Silico)

Uniprot: The Glucocorticoid Receptor ligand binding domain (GR-LBD) was identified under the entry number P04150-GCR_Human, gene NR3C1.

RCSB-PDB:The specific mutations described in He, Y et al., (2014) was retrieved from Xstal structure with the entry number 4P6X.

GenScript: Used for rare codon analysis.

Twist Bioscience: Used for the codon optimization tool.

PCR linearization of vector and cloning

A plasmid can be amplified by using PCR specific primers designed to exclude a specific region and, in this way, we will obtain a linear copy of the cloning vector to use, pET pET-28a transformed into TOP10 competent E. coli strains. The exclusion of this section of the vector produces overhangs that were considered in the design of our gBlock, and that way include our desired coding sequence into the cloning vector pET-28a.

The resulting PCR linearization products contains the coding sequence of our protein of interest...Read more content to avoid excessive length.

The final PCR products of the linearization were cleaned, the concentration and quality measured using Nanodrop. The PCR products were then prepared for the Gibson assembly.

Gibson Assembly (Insertion of GBlock)

Gibson assembly was utilized to clone the gBlock, containing GR-LBD and His-tag, into the linearized vector obtained in the PCR linearization step. The resulting circularized vector was then to be used further to transform competent BL21(DE3) expression E. coli bacteria strain.

The Gibson Assembly master mix is based on three different enzymes withing a single buffer mixture to improve accuracy and efficiency. The enzymatic activities consist of:

  • Exonuclease activity: Creates single-strand DNA overhangs from the linearized double stranded cloning plasmid pER-28a that can be anneal to complementary single-stranded complementary overhangs from our gBlock.
  • DNA polymerase activity: Incorporates nucleotides to “fill” the gaps after the single strand DNA and our gBlock has been annealed.
  • Taq DNA activity: Covalently joins the annealed complementary DNA fragments, removing any nicks and creating a contiguous DNA fragment between the cloning vector and our gBlock.

The constructs were verified by sequencing and then were transformed into Top10 E. coli competent cells to be cloned.

Colony PCR and Gradient PCR was also performed into the resulting cloned cultures to confirm that we successfully inserted our gBlock into the cloning strains.

Transformation of BL21(DE3)

Bacterial transformation is used in molecular cloning to produce multiple copies of a DNA molecule, in our case the plasmid carrying our gBlock, and its subsequent expression. For our experiment, we choose BL21(DE3) competent E. coli strain, as it is a suitable and strain for efficient protein expression.

We use Heatshock transformation method to introduce the cloning vector with our gBlock into the expression E. coli strain. After the induced stress, the cells were incubated at 37°C in an SOC recovery medium.

Culture preparation and induction

Small scale induction

We produce a small-scale culture to test the expression conditions of our construct and to decide what is the most optimal condition for protein production. The pre-culture corresponded 3 ml of LB-Kanamycin-50 inoculated with a single colony of our construct BL21DE3-pET28-GR. After overnight incubation at 37°C, a 1:100 dilution was done to control the growing process of the liquid culture. We decided to test the construct incubating the culture at 20°C and 37°C and inducing expression (By adding ITPG and Prednisolone) after incubations times of 30 minutes, hour hours and overnight.

Large Scale induction

The optimal conditions for production of our construct were determined on an incubation of 20°C overnight incubation. We produced a large-scale culture by making a pre-culture corresponding of 15 ml of LB-Kanamycin-50 inoculated with a single colony of our construct BL21DE3-pET28-GR. After overnight incubation at 37°C, a 1:100 dilution was done by adding 15 ml of the overnight culture in a 1 liter of LB-Kanamycin and incubating at 37°C until the ideal OD was obtained (Range between 0.4 and 0.7). The induced culture was then incubated at 20°C overnight before moving to protein purification.

Protein purification

Mechanical lysis and Nickel-NTA agarose beads purification

For the small-scale induction, we used bead beater as the mechanical lysis method and the proceed to purify the samples with Nickel-NTA agarose beads, due to the high-protein binding and affinity purification of His-tagged fusion proteins. After the lysis process, we will expect to obtain a purified concentration of our protein of interest in the soluble fraction of the purification.

Chemical lysis

We also included trials of chemical lysis using the B-PER Bacterial Protein Extraction Reagent (Thermo Scientific) method when we didn’t obtain a successful separation of our protein of interest into the soluble fraction of the Nickel-NTA purification. We used this process as a way to troubleshoot the mechanical lysis with the bead beaters, as this was not very efficient to release our protein of interest.

Mechanical lysis using French Pressure Cell Press

For the large-scale induction we used a French Pressure Cell Press (Thermo IEC) to have a complete lysis of a big volume culture with the same conditions. Setting the cell pressure between 18.000 to 20.000 as the established pressure used for E. coli.

His-Tag purification Chromatography

For the large-scale induction we used a Ni-NTA affinity purification of a bind-wash-elute procedure in the Fast Protein Liquid Chromatography (FPLC-AKTA chromatography system), using a HisTrap™ FF 5ml columns GE Healthcare) with a sepharose matrix pre-charged with nickel ions. The column was equilibrated in 5 column volumes (CV) Buffer A (Running Buffer for chromatography-See protocols) before the filtered supernant obtained in the mechanical lysis was loaded into the column. The column was then washed with 15 CV of Buffer A before the His-Tagged protein was eluted during a 0-100% gradient of Buffer B (Elution Buffer for chromatography-See protocols) over 15 CV.

Fractions from the chromatography are analysed on an SDS-Page gel and Western blot.

SDS-Page

Is a method used to separate proteins from a complex sample mixture, based on the size of the proteins. All the samples had to be denaturated before loading into the gel and be negatively charged to be separated by size while they move towards the positive probe in the SDS-Page apparatus.

The SDS-Page gels stained with Coomassie stain solution proofs the location of the protein of interest. Otherwise, SDS-Page gels were also used as the first step for Western Blotting.

Western blot

Is a standard technique used to identify specific proteins by exposing the physical membrane where the proteins bands were transferred to an antibody that will specifically target the protein of interest.

For our experiment, we wanted to confirm the presence of our protein of interest after the chromatography process by targeting the His-tag motif included in our Gblock with a specific antibody targeting it. This would be evidence that the results obtained on the purification process and SDS-Page screening corresponded to our resulting protein of interest.

Future perspectives

MST assay

The Microscale Thermophoresis Assay (MST) is biophysical assay that quantify the protein-ligand interaction by measuring the motions of molecules in microscopic temperature gradients that fluctuates upon ligand binding. This assay does not require high amounts of purified analyte samples and can be done directly in solution without any additional surface for mobilization. We want to proof the interaction between our obtained and purified GR-LBD protein with the ligand of interest, as a proof of concept that our construct works and can be used in further trials and experiments.

He Y, Yi W, Suino-Powell K, Zhou XE, Tolbert WD, Tang X, Yang J, Yang H, Shi J, Hou L, Jiang H, Melcher K, Xu HE. Structures and mechanism for the design of highly potent glucocorticoids. Cell Res. 2014 Jun;24(6):713-26. doi: 10.1038/cr.2014.52. Epub 2014 Apr 25. PMID: 24763108; PMCID: PMC4042175.

Protocols

Resuspend primers

Materials

  • Sterile distilled water or TE buffer
  • GR-Amp-F2 22.4 nmol
  • GR-Amp-R2 23.5 nmol
  • pET28-Linear-F 23.9 nmol
  • pET28-Linear-R2 26.5 nmol
  • pET28-seq-R 36.2 nmol

Procedure

Primer resuspension for stock

  1. Centrifuge the tube containing the primer powder to avoid any volume to not mix properly.
  2. For a stock solution of 100µM, the calculations are made following the nmol amount of each primer.
Primer with correspondent nmol Stock volume calculation
GR-Amp-F2 22.4 nmol 22.4nmol/(100µM) = 224 µL
GR-Amp-R2 23.5 nmol 23.5nmol/(100µM) = 235 µL
pET28-Linear-F 23.9 nmol 23.9nmol/(100µM) = 239 µL
pET28-Linear-R2 26.5 nmol 26.5nmol/(100µM) = 265 µL
pET28-seq-R 36.2 nmol 36.2nmol/(100µM) = 362 µL
pET28-seq-F 30.3 nmol 30.3nmol/(100µM) = 303 µL
  1. The calculated volume for each primer is the necessary volume of ddH2O or TE Buffer to reach a stock primer concentration of 100µM.
  2. Add the respective volume into each tube and vortex briefly until the solution is homogeneous.
  3. Store the stock primers at -20°C.

Primer resuspension for working solution

  1. Every primer solution was diluted in a 1:10 dilution ratio of the stock solution in new Eppendorf tubes.

V1 = (C2 * V2) / C1
(10µM * 10µL) / 0.1mM = 100µL
100µL - 10µL = 90µL

  1. Store the working solution in the freezer or fridge for as long as needed.

Preparation of the cloning vector pET-28a

Materials

  • Qiaprep Spin Miniprep Kit (QIAGEN)
  • pET-28a vector culture
  • 5x RxN Buffer
  • 10mM dNTPs
  • 10µM pET28-Linear-F
  • 10µM pET28-Linear-R
  • Template (1mg/µl)
  • Q5 polymerase
  • dd water

Procedure

Qiaprep Spin Miniprep Kit (QIAGEN)

  1. Distribute the 3ml culture of pET28 vector into two separate Eppendorf tubes.
  2. Centrifuge into pellet at 6800 x g for 3 minutes at room temperature.
  3. Remove supernatant in a safety disposal container, combine and resuspend the pellet in 250µl of Buffer P1.
  4. Add 250µl of Buffer P2 and incorporate it by inverting tube 4-6 times.
  5. After about 4-5 minutes 350µl of N3 was incorporated it the same way.
  6. Centrifuge for 10 minutes at 12.300 x g.
  7. Apply all the supernatant to the QIAprep 2.0 spin column by pipetting, and centrifuge for one minute at 12.300 x g.
  8. Discard the flowthrough, add 500µl of Buffer PB to the column by pipetting and centrifuge for one minute at 12.300 x g.
  9. Discarded the flowthrough, add 750µl of Buffer PE by pipetting and centrifuge for one minute at 12.300 x g.
  10. Discard the flowthrough and dried the column by centrifugation in one minute at 12.300 x g.
  11. Transfer the column to a collection tube, add 50µl of water to the center of the column and let it stand for one minute before eluting the product by centrifugation in one minute at 12.300 x g.

PCR

  • PCR for up concentration of the cloning vector.
PCR solution (100 µl) Required volume
5x RxN Buffer 10 µl
10mM dNTPs 1 µl
10µM pET28-Linear-F 2.5 µl
10µM pET28-Linear-R 2.5 µl
Template (1mg/µl) 0.5 µl
Q5 polymerase 0.5 µl
dd water 83 µl
  • Thermocycler program used for the PCR

Thermocycler program (28 cycles)

Step Temperature (°C) Time
Denaturation 98 °C 0:10
Q5 Anneling 58 °C 0:30
Extending 72 °C 1:40
Final extending 72 °C 2:00

Electrophoresis gel

  1. Combine 0.4g agarose and 50ml TBE Buffer in an Erlenmeyer flask and distribute the agarose granules by swirling the flask.
  2. Promptly put the flask in the microwave and periodically swirl the flask to prevent any regions of higher agarose concentration forming.
  3. Once solution is boiling, cool the flask down under flowing water.
  4. When tempered, add 5µl of GelRed to the solution.
  5. Poured the solution into the previously prepared vessel, and let it set for 40 minutes.

Gibson Assembly

Introduction

Gibson assembly was utilized to clone the Gblock, containing the glucocorticoid gene, into the linearized vector which had been formed from the PCR of the circularized vector. The resulting circularized vector was then to be used further to transform competent TOP10 E. coli cells.

Materials

  • Gblock pET-28-His-GR solution (100 ng/µl) Integrated DNA Technologies (Ref# 239413871)
  • Gibson Assembly Master Mix (2X) (NEBio #E2611)
  • Vector solution pET-28a(+) (126.79 ng/µl) 9 µl (pET-28a)

Procedure

  1. Take 1 µl of the stock Gblock solution (100 ng/µl) to make a 1x working solution by adding it to a new Eppendorf tube with 99 µl of sterilized distilled water.
  2. The quantity of the Gblock needed for the Gibson assembly mix is calculated using the NEBioCalculator: https://nebiocalculator.neb.com/#!/ligation with the following input:
    • Insert DNA length: 855 bp
    • Vector DNA length: 5 kb
    • Vector DNA mass: 120 ng (estimate from 126.79 ng/µl provided by NanoDrop)
    Total volume determined = 1.43µl
  3. Gibson assembly mix consists of mixing the following reagents:
    4. Gibson assembly mix Required volume
    Gibson Assembly MasterMix (2x) 2.43µl
    Gblock pET28-His-GR 1.43 µl
    Expression vector pET28a (+) 1.00 µl
  4. Incubate the Gibson assembly at 50˚C for 1h.

Transformation of cells

  1. Add 4.86µl of Gibson assembly mix to 100µl of Top10 E.coli competent cells.
  2. Incubate in ice for 30 minutes.

Colony PCR

Introduction

We ran the colony PCR to verify if the prepared colonies had successfully incorporated our sequence of interest, our constructed Gblock.

Materials

  • LB Broth medium
  • Kanamycin 100µg/ml
  • PCR Lysing buffer
  • PCR-strip tubes
  • Bacteria culture

Procedure

Agarose 0.8% gel

  1. Combine 0.4g agarose and 50ml TBE Buffer in an Erlenmeyer flask and distribute the agarose granules by swirling the flask.
  2. Promptly put the flask in the microwave and periodically swirl the flask to prevent any regions of higher agarose concentration forming.
  3. Once solution is boiling, cool the flask down under flowing water.
  4. When tempered, add 5µl of GelRed to solution.
  5. Pour the solution into the previously prepared vessel, and let it set for 40 minutes.

LB-Kan liquid culture medium

  1. Add 1 ml of Kanamycin 100µg/ml into 1L of LB broth culture medium.
  2. Keep refrigerated until further use.

PCR lysing buffer preparation

  1. Follow the instructions for the preparation of the buffer in a suitable container.
  2. Constantly stirring while adding the reagents.
PCR lysing buffer Required volume
dd H2O 1752 ml
10% Triton 200µl
1M Tris pH 8.0 40µl
500 mM EDTA 8µl
  1. Prepare 7 Eppendorf tubes with 3 ml of LB broth.
  2. Also prepare 7 PCR tubes with 30µl of PCR lysis.
  3. Transfer 7 independent colonies to the tubes with a loop into the PCR tubes, dipping the colony first into the corresponding LB Broth Eppendorf tube in order to keep a matching culture growing.
  4. An 8th colony was picked from the original culture previously processed on miniprep, as a control.

PCR protocol

  1. Made the PCR MasterMix following the presented instructions:
PCR MasterMix (200 µl x 8 tubes) Required volume
Q5 5x Buffer 40 µl
10mM dNTPs 4 µl
10µM pET28-Linear-F 10 µl
10µM pET28-Linear-R 10 µl
Template (1mg/µl) 4 µl (0.5 per tube)
Q5 polymerase 2 µl
dd water 130 µl
  1. Add 24.4 µl PCR MasterMix into each PCR strip tube first and then add 0.5µl DNA template.
  2. Follow the thermocycler program to run the PCR:
Step Temperature (°C) Time
Initial denaturation 95 °C 0:30
Denaturation 95°C 0:30
Q5 Anneling 50 °C 1:00
Extending 68 °C 1:00
Final extending 68 °C 5:00
  1. Add 3µl of 6x Buffer dye to the PCR product and then pipetted 3µl of the mixture to the well in the gel. Repeat the same procedure with all tube samples.
  2. Ran the gel for 40 minutes at 100V.

Gradient PCR

Introduction

We ran the GradientPCR to verify if the prepared colonies had successfully incorporated our sequence of interest, our constructed Gblock. Also, while assessing the optimal temperature of annealing for our PCR Protocol.

Materials

  • LB Broth medium
  • Kanamycin 100µg/ml
  • PCR Lysing buffer
  • PCR-strip tubes
  • Bacteria culture

Procedure

Agarose 0.8% gel

  1. Combine 0.4g agarose and 50ml TBE Buffer in an Erlenmeyer flask and distribute the agarose granules by swirling the flask.
  2. Promptly put the flask in the microwave and periodically swirl the flask to prevent any regions of higher agarose concentration forming.
  3. Once solution is boiling, cool the flask down under flowing water.
  4. When tempered, add 5µl of GelRed to the solution.
  5. Poured the solution into the previously prepared vessel, and let it set for 40 minutes.

LB-Kan liquid culture medium

  1. Add 1 ml of Kanamycin 100µg/ml into 1L of LB broth culture medium.
  2. Keep refrigerated until further use.

PCR lysing buffer preparation

  1. Follow the instructions for the preparation of the buffer in a suitable container.
  2. Constantly stirring while adding the reagents.
PCR lysing buffer Required volume
dd H20 1752 ml
10% Triton 200µl
1M Tris pH 8.0 40µl
500 mM EDTA 8µl
  1. Prepare 7 Eppendorf tubes with 3 ml of LB broth.
  2. Also prepare 7 PCR tubes with 30µl of PCR lysis.
  3. Transfer 7 independent colonies to the tubes with a loop into the PCR tubes, dipping the colony first into the corresponding LB Broth Eppendorf tube in order to keep a matching culture growing.
  4. An 8th colony was picked from the original culture previously processed on miniprep, as a control.

PCR protocol

  1. Made the PCR MasterMix following the presented instructions.
PCR MasterMix (200 µl x 8 tubes) Required volume
Q5 5x Buffer 40 µl
10mM dNTPs 4 µl
10µM pET28-Linear-F 10 µl
10µM pET28-Linear-R 10 µl
Template (1mg/µl) 4 µl (0.5 per tube)
Q5 polymerase 2 µl
dd water 130 µl
  1. Add 24.4 µl PCR MasterMix into each PCR strip tube first and then add 0.5µl DNA template.
  2. Follow the thermocycler program to run the PCR.

Thermocycler program (30 cycles)

Step Temperature (°C) Time
Initial denaturation 95 °C 0:30
Denaturation 95°C 0:30
Q5 Anneling 50 °C 1:00
Extending 68 °C 1:00
Final extending 68 °C 5:00
  1. Add 3µl of 6x Buffer dye to the PCR product and then pipetted 3µl of the mixture to the well in the gel. Repeat the same procedure with all tube samples.
  2. Ran the gel for 40 minutes at 100V.

Enzyme Digestion

Materials

  • Linear vector circular plasmid
  • Hind III
  • Control circular plasmid uncut
  • PCR-strip tubes
  • Bacteria culture

Procedure

Initial verification of backbone plasmid

  1. We ran electrophoresis using linear vector sample circular plasmid + Hind III and control circular plasmid uncut.
Sample Group Composition
Sample of linear vector 1.5 µl from concentration of 126 µg/µl linear vector + 8.5 µl of water
Sample of cut circular plasmid 4 µl from 40 µg/µl circular plasmid + 4 µl of water + 1 µl CutSmart Buffer + 1 µl 20 k/µl Hind III
Sample of control circular plasmid 4 µl C. plasmid 40 µg/µl + 5 µl water + 1 µl CutSmart Buffer

Enzyme Digestion

  1. Incubate the cut and control C-plasmid samples in a PCR machine with a heat block temp 37°C and lid temperature of 38°C for 15 minutes.
  2. After incubation, add 2 µl of GelRed dye to each sample: Linear vector, cut C. Plasmid, and control C. plasmid.
  3. Take 6 µl of each and add to 0.8% agarose gel with 1.5 µl 1kb ladder.
  4. Follow Culture PCR protocol to run PCR process for the seven independent colonies from our cryostock of pET27-GR.
  5. Inoculate the 7 colonies from our cryostock.
  6. After electrophoresis, all 7 colonies showed a strong band at ~1000 kb, indicating that all 7 colonies had incorporated the vector.

Transformation of BL21(DE3)

Materials

  • BL21(DE3) Competent E. coli
  • 2 PCR colony plasmid products (33ng/ µl)
  • LB-Kanamycin Agar plate
  • SOC medium
  • 50% glycerol

Procedure

Transformation of BL21(DE3) competent E.coli

  1. Thawing of two tubes of PCR colony plasmids.
  2. Thawing of BL21(DE3) Competent E. coli tube.
  3. After completely thawed, keep both tubes on ice.
  4. Add 100 µl of BL21 in a new Eppendorf tube, and then add 1 µl of plasmid.
  5. Incubate on ice for 30 minutes.
  6. Heat shock at 42°C for 90 secs in water bath.
  7. Transfer tube to ice for 2 minutes.
  8. In a clean security bench, add 900 µl of SOC to the tube.
  9. Incubate at 37°C 880 RPM for 1 hour.
  10. Plate 100 µl on a pre-warmed kanamycin plate at 37°C.
  11. Incubate at 37°C overnight.
  12. Transfer to fridge the next morning.

Pre-culture for stock

  1. Get the BL21DE3 + pET28-GR plate and the LB-Kan culture media from the fridge, along with a 13 ml culture tube.
  2. Pipette 5 ml of LB-Kan media into the culture tube, in a clean security bench.
  3. Inoculate culture tube with a single colony from the plate using a loop.
  4. Place culture tube in the shaking incubator at 37°C, 200 rpm overnight.
  5. Place LB-Kan bottle and plate back in the fridge.

Cryo-Stock of protein expression strain BL21DE3-pET28-GR

  1. Add 500 µl of the overnight culture solution into a 2 ml Screw cap tube.
  2. Add 500 µl of 50% glycerol and mix well by inversion.
  3. Label the tube clearly and place it at -80°C.

Culture Preparation and Induction

Materials

  • Culture plate with BL21DE3-pET28-GR colonies
  • LB broth kanamycin-50 liquid medium
  • 1 M IPTG
  • 2.88 mM prednisolone
  • Centrifuge Avanti J-26S XP, Beckman Coulter
  • 1x PBS

Procedure

Pre-culture Liquid Solution

  • Before starting, have a freshly streaked culture plate of BL21DE3-pET28-GR colonies. If possible, prepare the plate a day before the culture preparation and leave it incubating at 37°C overnight.
  • Pipette 15 ml of LB-Kan into a 100 mL flask in the security cabinet bench.
  • Inoculate the culture flask with a single colony from the plate using a loop.
  • Place culture tube in shaking incubator at 37°C and 200 rpm overnight.

Dilution of the Culture and Induction

  • 1:100 dilution by taking 15 ml of the overnight pre-culture and adding it to 1L of LB-Kan in a suitable container. Shake softly with each addition.
  • Separate the inoculated solution into 500 mL portions into two 2L conical flasks to fit the incubator and to allow optimal bacterial growth.
  • Incubate the cultures at 37°C 220 rpm until reaching the optimal Optical Density (OD)
    • Optimal OD score = 0.4 – 0.7
    • Considering the pre-culture volume suggested, it is expected that the culture will reach the optimal OD score after 2 hours and 2 hours 50 minutes. Check constantly during the period to stay within the optimal range.
  • Once the culture has reached an optimal OD, add 12.5 μl of IPTG and 177 μl of prednisolone to each flask. Also, add a drop of an anti-foaming agent.
  • Incubate the flask at 20°C overnight.

Centrifugation and Storage

  • After the overnight incubation, transfer the liquid cultures to a 1-liter container suitable for use in the Centrifuge Avanti J-26S XP.
  • Load the container and balance the centrifuge with a container filled with water until reaching the approximate weight of the container with the liquid culture and set the following conditions on the centrifuge.
Rotor ID Speed Time Temperature
JLA 8.1000 5,000 x g 15 minutes 4°C
  • Discard the supernatant into containers with bleach for safe disposal.
  • Add 35 mL of PBS into the pellet to dissolve gently. Use the same pipette or vortex the container to resuspend and mix the PBS constantly.
  • Transfer the resuspended pellet to a 50 mL Falcon tube.
  • Centrifuge at top speed for 15 minutes.
  • Discard the supernatant and store the pellet in a freezer at -20°C.

SDS-Page Gel for Coomassie Blue Staining

Materials

  • SDS-Page apparatus
  • 1x SDS-Page running buffer
  • 4x SDS-Page loading buffer
  • Coomassie staining solution
  • Novex 4-20%, Tris-Glycine PlusWedgeWellTM Gel, Invitrogen (Ref XP04202BOX)
  • PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (Catalog Num=26619)

Procedure

Sample Preparation

  • Sample of interest is aliquoted in 20 μl volumes in new working Eppendorf tubes to have a precise calculation of the amount of loading buffer and samples that will be loaded into the gel.
  • Add 5 μl of 4x SDS-Page loading buffer to each working tube.
  • Incubate samples at 75°C for 5 minutes.

Gel Loading

  • Prepare SDS-Page apparatus and load the Novex 4-20%, Tris-Glycine PlusWedgeWellTM Gel into the apparatus.
  • Fill the SDS-Page apparatus with 1x Running buffer until the gel and the bottom of the apparatus is completely covered.
  • Load the first well with 3 μl of the Prestained Protein ladder.
  • Load 5 μl of the working samples to fill up the rest of the wells.
  • Run at 225 V for approximately 40-45 minutes.

Coomassie Stain

  • Put the gel on a separate clean container or recipient, wash shortly with a bit of distilled water and then discard the liquid.
  • Cover the gel with the Coomassie staining solution (approximately 25 ml) and put the container in a moving rack for 10 minutes.
  • Pour the staining solution off into a chemical container, wash the gel with distilled water and then discard it.
  • Cover the gel with destaining solution and put the container in a moving rack for 1 hour.
  • Pour the destaining solution off into a chemical container, wash the gel with distilled water and then keep it covered until visualization.
  • The gel can be kept covered with distilled water overnight for better resolution during the imaging process.

Protein Purification

Materials

  • Lysis buffer
  • Running buffer
  • Elution buffer
  • DNAse
  • French Pressure Cell Press, Thermo EIC
  • Centrifuge Avanti J-26S XP, Beckman Coulter
  • Puradisc syringe filters of 0.45 μm and 0.20 μm
  • HisTrapTM FF 5ml column, GE Healthcare
  • FPLC-AKTATM chromatography system, GE Healthcare

Procedure

Sample Preparation

  • Add samples of bacterial culture into a 50 ml falcon tube. Record the empty and filled weights of the tube to have an approximation of the culture weight for the next steps.
  • Centrifuge at top speed for 15 minutes.
  • Discard the supernatant completely and weigh the approximate weight of the pellet.
  • Add 30 mL of lysis buffer and 50 μl of DNAse per mL of lysis buffer (1500 μl) to the resulting pellet. The final volume of the working sample must be a maximum of 35 mL.
  • Put the falcon tube on the roller mixer for half an hour.

Mechanical Lysis

  • Set the piston in the security location and add glycerol to the moving sections for better movement.
  • Start the lysis by loading the sample into the piston by changing the ratio selector to allow a pressure difference. Set the cell pressure between 18,000 to 20,000 as the established pressure used for E. coli, Piston high ratio between 1150 to 1280.
  • Repeat the lysis three times. Be aware of the pressure levels at any step of the process.
  • Release the pressure completely at the end of the run.
  • Take 1 mL of the resulting lysed solution in a separate Eppendorf tube. This sample will be used as a reference of an intermediate stage and to check the presence of the protein of interest at stages of the purification process with an SDS-Page gel. Label accordingly to keep track of the stage it represents.

Centrifuge Protocol

  • Load the resulting lysed sample into the centrifuge and set the following conditions for the centrifuge process:
Rotor ID Speed Time Temperature
JA 25-50 30,000 x g 30 minutes 4°C
  • Take 1 mL of the resulting solution after the centrifugation in a separate Eppendorf tube. This sample will be used as a reference of the intermediate stage and to check the presence of the protein of interest at stages of the purification process with an SDS-Page gel.
  • Take a section of the pellet with a loop and dilute it in 1 mL of PBS. This sample will be used as a reference of the intermediate stage and to check the presence of the protein of interest at stages of the purification process with an SDS-Page gel.
  • The resulting supernatant is filtered twice using the 0.45 μm and 0.20 μm syringe filters, respectively.
  • Attach a plastic syringe to the filters and pour the supernatant to pass across the filter into a new sterilized falcon tube.
  • Take 1 mL of the resulting solution from the filtrate process. This sample will be used as a reference of the intermediate stage and to check the presence of the protein of interest at stages of the purification process with an SDS-Page gel.

His-Tag purification Chromatography

  • Equilibrate the HisTrapTM FF 5ml column with 5 column volumes (CVs) or 25 mL of the running buffer.
  • Load the filtered supernatant onto the column.
  • Wash the column with 15 CVs of the running buffer.
  • Elute the protein by applying a 0-100% gradient of the elution buffer over 15 CVs.
  • Acquired fractions are to be analyzed using SDS-PAGE and Western blot.

Western Blot

Materials

  • Blotting Buffer
  • Blocking Buffer
  • Transfer Buffer
  • TBST
  • His-Tag Horseradish Peroxidase-conjugated Antibody, Biotechne (Product # MAB050H)
  • PVDF Transfer Membrane, 0.45 μM, for Western Blotting applications. Thermo Scientific (Product #88518)
  • Novex 4-20%, Tris-Glycine PlusWedgeWellTM Gel, Invitrogen (Ref XP04202BOX)
  • ECL TM Prime Western Blotting Detection Reagents, Cytiva (Ref RPN2232)
  • PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (Catalog Num=26619)
  • Methanol
  • Filter paper

Procedure

SDS-Page

  1. Load protein into the well of the SDS-Page gel, along with the PageRuler™ Protein Ladder.
  2. Run the gel at 190 V until the bands reach the end of the gel (approximately 40-50 minutes).

Western Blotting

  1. Cut the membrane and the filter paper sheets to the approximate size of the gel.
  2. Activate the membrane with methanol for 1 minute and rinse it afterwards with transfer buffer before preparing the stack.
  3. Stack the membrane in the following order:
    • Three sections of Filter paper - PVDF - SDS-Page Gel - Three sections Filter Paper
  4. Wet all the sections of the stack with transfer buffer after assembly.
  5. Place the stack in the TE 70x semi-dry transfer unit from Hoefer and start electrophoretic transfer with a voltage of 30 V for 1 hour.
  6. Block the membrane for 1 hour at room temperature or overnight at 4°C.
  7. Incubate the membrane with the His-Tag Horseradish Peroxidase-conjugated Antibody for 1 hour at room temperature or overnight at 4°C.
  8. Wash the membrane with TBST 3 times, 5 minutes each time in a shaker.
  9. Remove the membrane with WB forceps, slightly absorb excess liquid with absorbent paper, and place the membrane on clean cling film.
  10. Depending on the size of the film, add 1 ml of ECL-prime working solution per 10 cm2 of the film in drops onto the membrane, ensuring that the solution covers the membrane evenly, and leave it for 5 minutes at room temperature.

Working ECL-prime = 1.5 ml Solution A (luminol) + 1.5 ml Solution B (peroxide)

  1. Remove the excess liquid with absorbent paper. Place the membrane between two pieces of cling film, then place the membrane into the chemiluminescence imager, and take pictures.

Buffer Recipes

1x Transfer buffer for western blotting

Introduction

Solution is used to transfer the separated proteins from the SDS-Page gel to a PVDF membrane to obtain a good visualization of the proteins of interest.

Reagents

1 L Transfer buffer Required volumes/weight
48 mM Tris base 5.76 g/L
39 mM Glycine 2.95 g
20% Ethanol 200 ml

Procedure

  • Add the Tris base into the 200 ml ethanol with distilled water.
  • Add the glycine and complete the desired volume with distilled water.
  • Keep refrigerated at approximately 4°C

4x SDS-Page loading buffer

Introduction

Used for preparing and loading protein samples into polyacrylamide gels for visualization and posterior analysis.

Reagents

15 ml 4X SDS-Page loading buffer Required volumes/weight
200 mM Tris-Cl (pH 6.8) 3 ml
400 mM DTT 925 mg
8% SDS 1.2 g
0.4% Bromophenol blue ~60 mg
40% Glycerol 6 ml

Procedure

  • Add the reagents into 15 ml Falcon tube.
  • The required volume of Bromophenol blue is an approximation, as it was added while checking the color of the buffer until it was sufficiently blue.
  • Keep at room temperature.

10x SDS-Page Running Buffer

Introduction

Used as the stock solution to prepare 1x Running Buffer, the electrophoresis buffer for the SDS-Page visualization procedure.

Reagents

1L 10x SDS-Page Running Buffer Required volumes/weight
0.2051 M Tris base 30.3 g/L
1.924 M Glycine 144.4 g/L
0.03467 M SDS 10 g/L

Procedure

  • Add 800 mL of distilled water in a 1L container
  • Add the Tris base to the solution while stirring. Then add the calculated volume of glycine and SDS next.
  • Keep stirring the solution for approximately an hour.
  • Complete 1 liter of volume by adding distilled water.
  • Keep at room temperature.

Coomassie staining solution

Introduction

Solution used for visualization of proteins in an SDS-Page gel and protein detection.

Reagents

1L Coomassie Stain Required volumes/weight
0.125% Coomassie blue R250 1.25 g/L
10% Acetic acid 100 mL/L
40% Ethanol 500 mL/L

Procedure

  • Dissolve the Coomassie blue R250 in 500mL ethanol while stirring.
  • Stir the solution for 3-4 hours until it has been homogenized. Filter the solution through Whatman filter paper to remove any insoluble material
  • Add the acetic acid
  • Complete the volume with distilled water until reaching the total volume
  • Keep at room temperature.

Coomassie Destaining solution

Introduction

Destaining agent important to remove the additional dye present in the gel and to obtain a clear background on the imaging of the SDS-Page gel

Reagents

1L Coomassie Destain Required volumes/weight
10% Acetic acid 100 mL/L
40% Ethanol 500 mL/L

Procedure

  • Add 500 mL of ethanol in 400 mL of distilled water
  • Add 100 ml of acetic acid to reach the total volume required.
  • Keep at room temperature.

Elution buffer for protein purification and chromatography

Introduction

One of the crucial buffers for affinity purification of proteins.

Reagents

1L Elution Buffer Required volumes/weight
20 mM NaPi pH 8.0 2.84 g/L
150 mM NaCl 8.77 g/L
500 mM Imidazole 34.04 g/Ltd>
10% Glycerol 100 ml/L
1 mM DTT 154.25 mg/L
10 μM Ligand 3.571 mL of 2.8 mM stock/L

Procedure

  • Add the powder reagents and the glycerol into a suitable container to be stirred
  • Add 800 mL of distilled water while stirring until the solution gets homogenize
  • Add the ligand and DTT last to avoid changing the temperature of the tubes while adding the rest of the reagents
  • Complete the total desired volume with distilled water
  • The resulting solution must be filtered and degassed before putting it in a sterilized suitable container to avoid adding bubbles into the chromatographer.
  • Keep refrigerated at approximately 4°C

Lysis Buffer

Introduction

Buffer used for protein purification protocols

Reagents

50 ml Lysis buffer Required volumes/weight
50 µM Prednisone 893 µl
10% Glycerol 100 mL (99.5% glycerol)
50 mM Tris 2.5mL of 1M Tris
400 mM NaCl 4 mL of NaCl (3M)
0.5% Chaps 250 mg

Procedure

  • Add the corresponding reagents on a 50 mL falcon tube.
  • Put the falcon tube on a tube roller mixer for approximately 30 minutes, until the buffer looks completely homogeneous.
  • Filter the buffer before pouring into a sterilized 50 mL falcon tube.
  • Kept on cold room at 4°C approximately

Blocking buffer for Western blotting

Introduction

Blocking buffer is necessary to prevent non-specific binding of antibodies to the membranes and allow a better visualization of the proteins of interest.

Reagents

50 mL Blocking buffer Required volumes/weight
5% Milk for Blocking Western Blots 2.5 g/50ml
1X TBST 50 mL

Procedure

  • Add the powder reagent to a 50 ml falcon tube before adding the total volume of TBST.
  • Put the falcon on a tumbler for approximately half an hour or until the buffer is completely homogeneous.
  • Keep refrigerated at approximately 4°C

Running buffer for chromatography

Introduction

One of the crucial buffers for the separation of proteins during the chromatography process

Reagents

1L Running buffer Required volumes/weight
20 mM NaPi pH 8.0 2.84 g/L
500 mM NaCl 29.22 g/L
50 mM Imidazole 3.4 g/L
10% Glycerol 100 ml/L
1 mM DTT 154.25 mg/L
10 μM Ligand 3.571 mL of 2.8 mM stock/L

Procedure

  • Add the powder reagents and the glycerol into a suitable container to be stirred
  • Add 800 mL of distilled water while stirring until the solution gets homogenize
  • Add the ligand and DTT last to avoid changing the temperature of the tubes while adding the rest of the reagents
  • Complete the total desired volume with distilled water
  • The resulting solution must be filtered and degassed before putting it in a sterilized suitable container to avoid adding bubbles into the chromatographer.
  • Keep refrigerated at approximately 4°C

TBST for Western blotting

Introduction

Wash buffer for the PVDF membrane after the incubation process

Reagents

500 mL TBST Required volumes/weight
25 mM Tris Base 1.51 g/50mL
150 mM NaCl 4.38 g/50ml
0.1% Tween20 detergent 0.5ml/50mL

Procedure

  • Add the reagents to an initial volume of 250 ml of distilled water to container suitable for stirring.
  • Complete desired volume with distilled water.
  • Keep refrigerated at approximately 4°C