Team:IISER Tirupati/Protocols

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Experiments
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PROTOCOLS

  1. Gradient PCR:
    Reaction Mixture:
    • Template - 5-10 ng (for plasmids), 20-100 ng (for gDNA)
    • 10 uM Forward Primer : final concentration - 500 nM
    • 10 uM Reverse Primer : final concentration - 500 nM
    • 2X Q5 Master Mix : final concentration - 1X
    • Dd H2O - upto final volume

    Temperature Profile:
    • Initial Denaturation 98°C: 1-2 min
    • Denaturation 98°C: 15 sec Annealing (Set gradient from Tm-10 to Tm+2°C): 20 sec Extension 72°C: Depends on Amplicon length (30 sec for 1 kb)
    • Final Extension 72°C: 10 min
    • Hold 4°C

    Run the PCR products in 0.8% Agarose Gel to check the right annealing temperature (the temp in which the PCR gives a clear band with minimum non-specificity)

  2. PCR Amplification protocol:
    Reaction Mixture:
    • Template - 5-10 ng (for plasmids) 20-100 ng (for gDNA)
    • 10 uM Forward Primer : final concentration - 500 nM
    • 10 uM Reverse Primer : final concentration - 500 nM
    • 2X Q5 Master Mix : final concentration - 1X
    • dd H2O - upto final volume

    Temperature Profile:
    • Initial Denaturation 98°C: 1-2 min
    • Denaturation 98°C: 15 sec Annealing (From Gradient): 20 sec Extension 72°C: Depends on Amplicon length (30 sec for 1 kb)
    • Final Extension 72°C: 10 min
    • Hold 4°C


  3. SOEing PCR protocol::
    Reaction Mixture:
    • Templates - the amounts (in mole) of the fragments (used to SOE) have to be same
    • 10 uM Forward Primer : final concentration - 500 nM
    • 10 uM Reverse Primer : final concentration - 500 nM
    • 2X Q5 Master Mix: final concentration - 1X
    • dd H2O - up to final volume

    Temperature Profile:
    • Initial Denaturation 98°C: 1-2 min
    • Denaturation 98°C: 15 sec Annealing (Higher Temperature for SOEing of the fragments): 25 sec Extension 72°C: Depends on Amplicon length (30 sec for 1 kb)
    • Extension 72°C: 5 min
    • Denaturation 98°C: 1 min
    • Annealing (Lower Temperature for the terminal primers to bind): 20 sec
    • Extension 72°C: Depends on Amplicon length (30 sec for 1 kb)
    • Final Extension 72°C: 10 min
    • Hold 4°C

  4. Colony PCR:
    Reaction Mixture:
    • Template - Single Colony in 5 ul ddH2O
    • 10 uM Forward Primer : final concentration - 500 nM
    • 10 uM Reverse Primer : final concentration - 500 nM
    • 10X Taq Buffer - final concentration - 1X
    • Taq Polymerase - 1 unit for each 50 ul reaction
    • dNTPs - final concentration - 200 uM
    • dd H2O - upto final volume

    Temperature Profile:
    • Initial Denaturation 98°C: 5 min
    • Denaturation 98°C: 15 sec Annealing (Tm-5)°C: 20 sec Extension 68°C: Depends on Amplicon length (1 min for 1 kb)
    • Final Extension 68°C: 10 min
    • Hold 4°C

  5. Restriction Digestion (Both Vector and Insert):
    Reaction Mixture:
    • Template - Enough to do Ligation
    • 10X Buffer (Suitable NEB RE Buffer): final concentration - 1X
    • Restriction Enzyme 1: 10 units for 1 ug of DNA
    • Restriction Enzyme 2: 10 units for 1 ug of DNA
    • ddH2O: up to final volume

    Temperature Profile:
    • Incubation 37°C: 150-180 mins
    • Heat Inactivation: Depends on Enzyme (65°C/80°C)
    • Hold 4°C

    After Digestion, PCR Purification for Insert and Gel Extraction for Vector

  6. CIP Treatment (For Vector only):
    Reaction Mixture:
    • Template: Enough to do ligation
    • 10X CutSmart: final concentration - 1X
    • CIP: 10 units for 1 ug of DNA ends
    • ddH2O: up to final volume

    Temperature Profile:
    • Incubation 37°C: 150-180 mins
    • Heat Inactivation: Depends on Enzyme (65°C/80°C)
    • Hold 4°C

  7. Ligation:
    Reaction Mixture:
    • Vector: 20-50 ng
    • Insert: 3-7 times more than vector
    • 10X Ligase Buffer: final concentration - 1X
    • T4 Ligase: 10 units for 1 ug of DNA ends
    • ddH2O: up to final volume

    Temperature Profile:
    • Incubation 16°C: Overnight or, Room Temperature: 2-3 hrs.
    • Heat Inactivation: 65°C
    • Hold 4°C

  8. Transformation:
    Materials:
    • Resuspended DNA to be transformed
    • Ligated Product (1 - 100 ng - Depending on the efficiency of the competent cells)
    • Competent Cells (50µl per sample)
    • 1.5 mL Eppendorfs
    • SOC Media (950 µL per sample)
    • Petri plates with LB agar and antibiotic (2 per sample)

    Equipment
    • Floating foam tube rack
    • Ice & ice bucket
    • Lab Timer
    • 42°C water bath
    • 37°C incubator (Both with shaker and static)
    • Sterile spreader or glass beads
    • Pipettes and Tips (10 µl, 20 µl, 200 µl recommended)
    • Microcentrifuge

    Method
    • Thaw competent cells stored in -80°C on ice for 20-30 mins.
    • Remove antibiotic plates and SOC media from 4°C and incubate it to attain room temperature.
    • Mix about 1-100 ng of plasmid DNA with 50 ul of competent cells (depending on efficiency) in a micro-centrifuge tube. Gently mix by flicking the tube a few times.
    • Incubate the mixture on ice for 30 mins. Meanwhile, prepare a water bath (heat block also works!) at 42°C.
    • Give heat shock at 42°C for 45 sec.
    • Put the tubes back on the ice for 15 mins.
    • Add 950 ul of SOC media to the mixture and incubate it at 37°C and 200 rpm for 1 - 1:30 hours.
    • Centrifuge at 4000g for 5 mins and resuspend the pellet in 100 uL of LB.
    • Plate the bacteria on the antibiotic plates and incubate the plates for 12-15 hrs at 37°C. Check for colonies.

  9. Competent cell preparation:
    Materials:
    • LB plates
    • 50 ml Falcon tubes
    • 1.5 ml microfuge tubes
    • 1 L flask
    • 85 mM CaCl2 + 15% glycerol
    • DH5-alpha cells and BL21 cells
    • liquid nitrogen
    • Mili Q water
    • ice (lots of it! keep the ice-machine on)
    • Glycerol
    • SOC (Super Optimal broth with Catabolite repression)
    • LB media plus 20 mM Glucose (Add sterile glucose after autoclaving)

    Procedure:
      Everything should be sterile and done under cold conditions, chill all apparatus beforehand
    • Spread an LB plate without antibiotic, with the bacteria of interest and incubate for 16 hours till isolated colonies 2-3 mm in diameter are obtained
    • Stocks preparation
    • 1 M glucose stock 50 ml - 5 g in 45 ml water, dissolve, make volume to 50 ml
    • 1 M 11.1 g of anhydrous salt in 100 ml water (for 0.1 M 2 ml in 18 ml of water)
    • 1 M MgCl2- 9.52 g of anhydrous salt in 100 ml of water (for 0.1 M 2 ml in 18 ml of water)
    • Make 200 ml of LB in 196 ml of water, autoclave and add 4 ml of glucose stock to get 20 mM of glucose finally = rough SOC (actually SOC also has MgCl2 and MgSO4 along with KCl)
    • Storage in 2 ml 85 mM calcium chloride + 15% glycerol for 50 ml of culture (make 10 ml using 1.5 ml glycerol + 0.85 ml CaCl2 stock + 7.65 ml water)
    • Pick a colony and inoculate in 10 ml of SOC and let it grow (primary culture)
    • Inoculate 100 ml of secondary culture to an OD of 0.1 or 0.2 and let it grow till 0.6-0.8 OD
    • Divide the culture into two 50 ml falcons
    • Cool them on ice for 30 mins
    • Spin down at 3000 rpm for 10 mins at 4 degrees
    • Resuspend in 20 ml of 0.1 M CaCl2 and keep on ice for 20 mins
    • Repeat steps 7 and 8
    • Finally, spin down the cells and resuspend in 2 ml of 85 mM CaCl2 + 15% glycerol
    • Aliquot out 50 ul of the suspension into eppendorfs (40 eppendorfs)
    • Snap freeze using liquid nitrogen
    • Store at -80°C

  10. Plasmid Isolation:
    Using GeneJET plasmid miniprep kit.

    Pick a single colony from the freshly streaked selective plate and inoculated in 5 mL of LB medium (for High copy number plasmid)/ 10 mL of LB medium (low copy number plasmid) supplemented with suitable Antibiotic. Incubate for 12-16 hrs at 37°C (shaking 200-250 r.p.m.). Each spin column can recover up to 20 ug of plasmid DNA.
    Materials
    • Resuspension solution (RNaseA added and stored at 4°C)
    • Lysis solution
    • Neutralization Solution
    • Wash solution (96-100% ethanol added)
    • Spin column
    • Collection tube (1.5 mL Eppendorf)
    • Nuclease Free Water (NFW) - at 60°C

    Procedure
    • Harvest the bacterial cells from the culture by centrifugation at 9000 g in a microcentrifuge for 5 min (2 min in the actual protocol) at room temperature. Decant the supernatant and remove all remaining medium. All the rest centrifuges should be carried out in a table-top microcentrifuge at >12000 g at room temperature
    • Resuspend the pelleted cells in 250 µL of the Resuspension Solution (from 4°C). The bacteria should be resuspended by pipetting up and down until no cell clumps remain.
    • Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. (Do not vortex the tube to avoid the shearing of chromosomal DNA).
    • As soon as the solution gets clear (about 2-3 mins after adding Lysis solution), add 350 µL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times until the lysate becomes cloudy. (the mixing should be done thoroughly and gently). (better to keep in ice for some time)
    • Centrifuge for 5 min to pellet cell debris and chromosomal DNA. (can be done for more time to get purer plasmid)
    • Transfer the supernatant to the supplied GeneJET spin column by pipetting. Avoid disturbing or transferring white precipitate to avoid protein contamination (Close the bag with GeneJET Spin Columns tightly after each use!)
    • Centrifuge for 30-40 sec. Discard the flow-through and place the column back into the same collection tube. (Do not add bleach to the flow-through for safety purpose)
    • Add 500 µL of the Wash Solution (ethanol added) to the GeneJET spin column. Centrifuge for 30-40 sec and discard the flow-through. Place the column back into the same collection tube.
    • Repeat the wash procedure (step 8) using 500 µL of the Wash Solution.
    • Discard the flow-through and centrifuge for an additional 2-3 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
    • Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube. Add 30-50 µL of hot nuclease-free water(60°C) to the centre of GeneJET spin column membrane to elute the plasmid DNA. (Incubate for 3-4 min after addition of water and then centrifuge for 5 min). Take care not to contact the membrane with the pipette tip.
    • Discard the column and store the purified plasmid DNA at -20°C.

  11. PCR Purification (Using GeneJET PCR Purification Kit):
    Materials:
    • GeneJET PCR Purification Kit (K0701, Thermo Fisher Scientific),
    • Ethanol 96-100%
    • Isopropanol
    • 3 M sodium acetate, pH 5.2 (may be necessary)
    • Microcentrifuge
    • 1.5 or 2 mL microcentrifuge tubes
    • Heating block

    Procedure:
    • Add a 1:1 volume of Binding Buffer to completed PCR mixture. Mix thoroughly. (Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.)
    • (Optional: if the DNA fragment is ≤500 bp) Add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly. Note. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.
    • Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. If the total volume exceeds 800 µL, the solution can be added to the column in stages. Avoid disturbing or transferring white precipitate to avoid protein contamination (Close the bag with GeneJET Spin Columns tightly after each use!). Centrifuge for 30-40 sec. Discard the flow-through and place the column back into the same collection tube. (Do not add bleach to the flow-through - Safety purpose)
    • Add 500 µL of the Wash Solution (ethanol added) to the GeneJET spin column. Centrifuge for 30-40 sec and discard the flow-through. Place the column back into the same collection tube.
    • Repeat the wash procedure (step 4) using 500 µL of the Wash Solution.
    • Discard the flow-through and centrifuge for an additional 2-3 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
    • Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube. Add 30-50 µL of hot nuclease-free water (60°C) to the centre of GeneJET spin column membrane to elute the plasmid DNA. (Incubate for 3-4 min after addition of water and then centrifuge for 5 min). Take care not to contact the membrane with the pipette tip.
    • Discard the column and store the purified plasmid DNA at -20°C.

  12. Gel Extraction Protocol (Using GeneJET PCR Purification Kit):
    Materials:
    • GeneJET PCR Purification Kit (K0701, Thermo Fisher Scientific),
    • Ethanol 96-100%
    • Isopropanol
    • 3 M sodium acetate, pH 5.2 (maybe necessary)
    • Microcentrifuge
    • 1.5 or 2 mL microcentrifuge tubes
    • Heating block

    Procedure:
    • Run the whole DNA sample in 1-1.2% Agarose gel long enough so that all the bands get separated enough to get clear individual bands.
    • Excise gel slice containing the desired DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice. Note. If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure by minimizing UV exposure time.
    • Add 1:1 volume of Binding Buffer to the gel slice (volume: weight).
    • Incubate the gel mixture at 50-60°C for 10 min until the gel slice is completely dissolved. Mix the tube by inversion every few mins to facilitate the melting process. Ensure that the gel is completely dissolved. Mix the solution thoroughly by pipetting up and down. (Check the colour of the solution. A yellow colour indicates an optimal pH for DNA binding. If the colour of the solution is orange or violet, add 10 μL of 3 M sodium acetate solution (pH 5.2) and mix. The colour of the mix will become yellow ).
    • For DNA fragments ≤500 and >10. If the DNA fragment is ≤500 bp, add 1 gel volume of 100% isopropanol to the solubilized gel. Mix thoroughly. If the DNA fragment is >10 kb, add 1 gel volume of water to the solubilized gel solution. Mix thoroughly.
    • Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. If the total volume exceeds 800 µL, the solution can be added to the column in stages. Avoid disturbing or transferring white precipitate to avoid protein contamination (Close the bag with GeneJET Spin Columns tightly after each use!). Centrifuge for 30-40 sec. Discard the flow-through and place the column back into the same collection tube. (Do not add bleach to the flow-through - Safety purpose)
    • Add 500 µL of the Wash Solution (ethanol added) to the GeneJET spin column. Centrifuge for 30-40 sec and discard the flow-through. Place the column back into the same collection tube.
    • Repeat the wash procedure (step 6) using 500 µL of the Wash Solution.
    • Discard the flow-through and centrifuge for an additional 2-3 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
    • Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube. Add 30-50 µL of hot nuclease-free water (60°C) to the centre of GeneJET spin column membrane to elute the plasmid DNA. (Incubate for 3-4 min after addition of water and then centrifuge for 5 min). Take care not to contact the membrane with the pipette tip.
    • Discard the column and store the purified plasmid DNA at -20°C.

Cell Culture


Reviving the cells:
  • Before beginning the procedure, all the materials required are brought to 37°C. by using a water bath.
  • The cells preserved at -80°C are brought to thawed by placing at 37°C for 45 sec.
  • The ice crystals, if any are dissolved by adding complete media in which the cells are to be cultured.
  • The solution can either be transferred directly to T-25 flask containing complete media or the solution can be centrifuged to obtain cell pellets followed by re-suspending in the media to be utilized for culturing.
  • Cells are cultured until they attain 80-90% confluency after which they are passaged.

Passaging the cells:
  • Before beginning the procedure, all the materials required are brought to 37°C by using a water bath.
  • Media from the culture flask is removed after the culture becomes confluent.
  • Add 2 ml of Phosphate Buffer Saline (PBS) and gently swirl to wash off the media.
  • Remove the PBS and add 1 ml of 0.25% trypsin EDTA solution (the volume depends on the type of culture dish). Keep the cells in the incubator at 37°C for 2-3 mins. Check under the microscope if the cells have started to dislodge. If the cells don’t come off gently tap the flask. If the problem still persists, incubate for more 1-2 mins and tap to check.
  • Remove the suspension using a pipette and add into a falcon which already contains some media.
  • Centrifuge at 200 g (for CRL-1790 cells) / 800 g (for HT29 cells) for 5 min at room temperature.
  • Resuspend the pellet in fresh media and distribute equally in culture dishes. (A cell count before distribution of cells to culture dishes is recommended)




HPLC

The lactate estimation was done using ‘SHIMADZU’ HPLC system. The data was recorded and analysed using ‘Lab solutions’ software. The estimation was done using Atlantis T18 column, which is a version of c18 column(4. 6*250 mm) and the column temperature was maintained at 25°C. The mobile phase used was Phosphate buffer (10 mM, pH 3) and acetonitrile with a ratio of 95:5. The flow rate was maintained at 1 ml/min, and the injection volume was 20 ul. The effluent was monitored at a wavelength of 210 nm.

  1. Buffer preparation (Ginjupalli Kishore et al. ,2013)
    Materials-
    • Potassium dihydrogen phosphate (KH2PO4) - 2. 72 g
    • Ortho-Phosphoric acid - 200-250 ul
    • Acetonitrile - 100 ml
    • Milli-Q water - 2 L
    The buffer (mobile phase) was prepared by dissolving 2. 72 g of KH2PO4 in 1900 ml of milli-Q water. Orthophosphoric acid was then added to adjust the pH to 3. The remaining solution was made up to 2000 ml using Milli-Q to make 10 mM phosphate buffer. The buffer solution was then filtered through a 0.22 um membrane filter and sonicated. Acetonitrile was also similarly sonicated. The mobile phase was prepared by 95:5 dilution of phosphate buffer and acetonitrile (100 ml of acetonitrile was added to 1900 ml of 10 mM phosphate buffer).
  2. Standard solution and standard curve preparation We proceeded with mM standard graph (since the concentration of lactate in cancer microenvironment is in millimolar range).
    For the preparation of a standard graph, known concentrations of lactate solutions were prepared using L-lactate solution (11. 4 M). Based on the molarity of the lactate solution,175 ul was added to 1825 ul of mobile phase to obtain 1 M concentration of lactate solution. Serial dilutions were then performed to obtain 5 mM, 10 mM, 25 mM, 50 mM, 75 mM and 100 mM concentrations of lactate solutions. The solutions were then filter sterilised using 0. 22 um PVDF filter. After filter sterilisation, the standard solutions were injected into HPLC and readings were taken.
  3. Sample preparation The HT-29 cells were grown in RPMI media, with 10% Fetal Bovine Serum (FBS). Since all the cells were grown in a medium containing 10% FBS, it was essential to filter the spent media before the assay, since FBS contains lactate dehydrogenase enzyme which will cleave the lactate to pyruvate. So we used a 3 kDa MWCO filter to remove the enzyme. The filtrate after centrifugation was collected and filter sterilised using 0.22 um PVDF filter.


GENERAL PROTOCOL
  • Preparation of buffer
  • Preparation of standard solution from known lactate concentration
  • Collection of cancer cell line spent media and centrifugation at 12000 rpm for 20 mins using 3 kDa filter
  • Filter sterilise the spent media and standard solutions using 0.22 um PVDF filter
  • Turn on HPLC machine, auto-purge for 15 minutes followed by column washing with water and buffer
  • Inject the standard solutions, 20ul each and run the samples
  • Note down the retention time, peak intensity and area for each sample
  • Plot the standard graph with area against concentration
  • Wash the column with the buffer for 30 mins
  • Inject and run the filter sterilised spent media, 20 ul
  • Observe the retention time, intensity and area
  • Spike with a known concentration of lactate to confirm the presence of lactate
  • Using a standard graph find the unknown concentration

The following protocol was used to characterise our sfGFP constructs:

  • Setting up primary culture: The cultures are first inoculated in 1 mL of SOC with required antibiotics (35 ug/mL- Chloramphenicol, 50 ug/mL- Kanamycin). The culture is covered with aluminium foil and kept for 2 hours in 37°C incubator. After this, centrifuge the cultures at 4000 g for 5 mins, remove supernatant and resuspend in 5 mL Minimal Media (M9) and antibiotics are added. The cultures are covered in foil and kept for 12-16 hours.
  • Setting up secondary culture: Centrifuge the primary culture, and resuspend the cells in 5 mL M9, add antibiotics. Check the OD of the culture in plate reader. Dilute the culture if necessary to get the OD of 0.1. Keep the culture for 5-6 hours.


‘Lactate Assay’ Protocol:

  • Once the OD of the culture reaches 0.4, add the culture to plate reader with appropriate blank (M9 + required lactate concentration) after induction with different concentrations of lactate. (0 mM, 0.01 mM, 0.03 mM, 0.06 mM, 0.1 mM, 0.3 mM, 0.6 mM, 1 mM, 3 mM, 6 mM, 10 mM, 13 mM, 16 mM, 20 mM). Our assays were done with 3 technical replicates.
  • Program the Gen5 software to shake the culture twice (Orbital- slow, split into 15:00 min and 12:30 min), read OD (at 600 nm) and then fluorescence at 480 excitation and 510 emission with 70 gain. These cycles can be set for 8-12 hrs, depending on the assay.
  • Analyse the data after completion of the program.




For characterising our IL-12 construct, we performed a western blot.

Western Blot:

Materials:
  • 15% SDS-PAGE gels
  • 5X Protein loading dye (containing 2-Mercaptoethanol)
  • Running buffer (3 g of Tris base, 14.4 g of glycine, and 1 g of SDS in 1 l of H2O. pH should be adjusted to ~8.3)
  • Transfer buffer (same as running buffer without the SDS and with 20% methanol)
  • PBST (1X PBS and 0.1% Tween 20)
  • Blocking solution (5% skimmed milk powder in 1X PBST)
  • Primary and secondary antibody
  • Staining solution (0.1% Coomassie Blue R250 in 10% glacial acetic acid, 50% methanol, and 40% water)
  • Destaining solution (10% glacial acetic acid, 40% methanol, 50% water)
  • Sponges, filter paper, PVDF membrane, ice pack, forceps
  • Pre-stained protein ladder
  • Electrophoresis tank, power source
  • Developing solutions for blot imaging


Sample preparation:
  • Grow induced and uninduced bacterial cultures at 37°C for 12 hours
  • Spin down the cultures at 3000 g for 5 mins
  • Transfer clear supernatant to a 10 kDa molecular weight cut-off concentrator and centrifuge at 4°C for till volume reduces to 500 ul
  • Resuspend the pellet in 1 ml PBS and check the OD
  • Dilute the pellets appropriately to obtain equal OD for both the samples
  • Take 80 ul of this resuspended pellet and add 20 ul of 5X loading dye and heat the sample on a heat block for 20 mins at 98 C
  • Similarly, take 80 ul of the concentrated supernatant and add 20 ul of the loading dye and heat
  • The samples can be stored or run on the gel


Western blotting procedure:
  • Load equal volumes of the pellets and equal volumes of the concentrated supernatants along with 5 ul of protein ladder. An SDS-PAGE should be run prior to the western blot to optimise the loading volumes such that there is no over/underloading.
  • Run the gel in the running buffer until the dye front reaches the end of the gel. Make sure that the buffer does heat too much (run the gel in 4°C or place the tank on ice).
  • As the gel is running, soak the sponges and filter papers in the transfer buffer
  • Cut a PVDF membrane corresponding to the gel size and put it in methanol. Make sure you do not touch the membrane. Handle with forceps.
  • Wash the membrane with transfer buffer. You can make a cut on one corner to remember the orientation of the membrane.
  • Once the gel is run, separate it from glass plates and wash it gently.
  • Next, make the arrangement in the following order: Anode face (red) - sponge - filter paper - membrane - gel - filter paper - sponge - Cathode face (black)
  • Put this setup in a tank containing prechilled transfer buffer and ice pack and run at 100 V for 2 hours. Make sure that the buffer does not heat up.
  • Once the run is complete, take the membrane and put it in a rectangular container and cover it with blocking solution and rock it for 1 hour. You should see the protein ladder on the membrane indicating that transfer has taken place.
  • To check the efficiency of transfer, stain the gel with staining solution for 45 minutes and destain for 2 hours.
  • After an hour, discard the blocking solution and add fresh blocking solution containing primary antibody with the dilution mentioned in its corresponding datasheet.
  • Incubate it overnight on a rocker at 4°C
  • Remove the primary antibody and store it in -20°C (it can be used ~3 times)
  • Give the membrane three 15 minute-washes with fresh blocking solution at room temperature
  • Add the secondary antibody in blocking solution and incubate at room temperature for 1 hour
  • Store this secondary antibody solution at -20°C for next use
  • Give the membrane three 15 minute-washes with PBST at room temperature
  • You can then image or store the blot in PBST at 4°C
  • For imaging, we used Peroxide-Luminol based solution as our secondary antibody was conjugated with HRP

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