ankyron-protocols

Ankyron™ Protocols

Suggested Ankyron™ Protocols

  1. Ankyrons™ in Flow Cytometry:
    1. Detection of extracellular target proteins
    2. Detection of cytoplasmic target proteins
    3. Detection of nuclear target proteins
  2. Ankyrons™ in Fluorescence Microscopy:
    1. Detection of extracellular target proteins in adherent cells
    2. Detection of intracellular (non-nuclear) target proteins in adherent cells
    3. Detection of nuclear target proteins in adherent cells
  3. Ankyrons™ in ELISA
  4. Ankyrons™ in Immunohistochemistry
  5. Ankyrons™ in Western blotting

*Please note that these protocols are starting point suggestions. They may require optimization for your specific application. We recommend titrating all reagents to determine the optimum quantities required.

Ankyrons™ in Flow Cytometry

Centrifuge Ankyron™ in a chilled microcentrifuge at 14,000 ×g for 5 min prior to use in all applications. This will remove protein aggregates that contribute to non-specific staining.

Allocate 0.3 – 1 × 105 cells per staining condition, in wells of a 96-well plate. The frequency of the target molecule on the cells of interest will dictate the number of cells required per staining condition and may require optimization.

All steps either on ice or at 4°C, unless otherwise stated, and use ice cold reagents. Low temperature prevents internalization of surface antigens, which could result in reduced fluorescence intensity. Protect all fluorescent reagents from light, to prevent photobleaching.

Detection of extracellular target proteins

Additional materials required: Wash Buffer (0.1 % sodium azide, 1 % fetal bovine serum, FBS in PBS); Fix Solution (1 % FBS, 1 % formaldehyde in PBS); Fixable Viability Dye.

1. Pre-conjugate V5-tagged Ankyron™ in PBS with anti-V5 Fluorotag for Ankyron™, 8 μl/test. We recommend trying an Ankyron™ titration range from 4 – 0.1 μg per test condition in the first instance. Allow 50 – 100 μL/sample. Shield from light and incubate at 4 °C for 30 min or overnight.

2. Wash the cells with an excess volume of Wash Buffer and resuspend them in the residual volume (~50 μL). Keep tubes chilled on ice for all subsequent steps, except where indicated.

3. Add the pre-conjugated V5-tagged Ankyron™ with anti-V5 Fluorotag for Ankyron™ and any additional labelled Ankyrons™ or antibodies required for staining to the cells. Mix well and incubate samples at +4 °C for 30 minutes.

4. Wash the cells with an excess volume of Wash Buffer.

5. Resuspend cells thoroughly in the residual volume before adding 100 μL Fix Solution. Store stained cells in Fix Solution at +4 °C in the dark until analysis.

Detection of cytoplasmic target proteins

Additional materials required: Wash Buffer (0.1% sodium azide, 1% FBS in PBS); Fix Solution (1% FBS, 1 % formaldehyde in PBS); Permeabilization buffer (e.g. BD Cytofix/Cytoperm); Fixable Viability Dye (optional).

1. Pre-conjugate V5-tagged Ankyron™ in PBS with anti-V5 Fluorotag for Ankyron™, 8 μl/test. We would recommend trying an Ankyron™ titration range from 4 – 0.1 μg per test condition in the first instance. Allow 50 – 100 μL/sample. Shield from light and incubate at 4 °C for 30 min or overnight.

If also staining for cell surface proteins, follow steps 1 – 4 in the section above, before permeabilizing cells ahead of detection of cytoplasmic target proteins.

2. Apply Fixable Viability Dye in PBS to the cells, as per the manufacturer’s instructions, and mix well. Incubate for 15 min at +4 °C. Typically, 1 in 1000 dilution, 100 μL/well is sufficient. (Optional, but recommended).

3. Wash cells, resuspend in residual volume and apply Fix/Perm solution, typically 100 μL/well, incubating for 20 min at +4 °C or as per the manufacturer’s instructions. During the incubation, prepare 1X Perm/Wash solution.

4. Wash cells in an excess of 1X Perm/Wash, centrifuging for up to 10 min at 400 x g for maximum cell recovery.

5. Add the pre-conjugated V5-tagged Ankyron™ with anti-V5 Fluorotag for Ankyron™ and any additional labelled Ankyrons™ or antibodies required for staining intracellular targets to the cells. Mix well and incubate samples at +4 °C for 30 minutes.

6. Wash cells twice in an excess of 1X Perm/Wash, centrifuging for up to 10 min at 400 x g for maximum recovery.

7. Resuspend cells in residual volume then add 100 μL/well 1X Perm/Wash Solution. Store samples in the dark at 4 °C until analysis.

8. Add the pre-conjugated V5-tagged Ankyron™ with anti-V5 Fluorotag for Ankyron™ and any additional labelled Ankyrons™ or antibodies, resuspended in 1X Perm Buffer, required for staining nuclear targets to the cells. Mix well and incubate samples at +4 °C for 30 minutes. The length and temperature of the incubation may require optimising, either at +4 °C or room temperature for 30 min or up to 16 h.

9. Wash cells in an excess of 1X Perm Buffer 2 – 3 times.

10. Resuspend cells in residual volume then add 100 μL/well Wash Buffer. Store samples in Wash Buffer in the dark at +4 °C until analysis.

Detection of nuclear target proteins

Additional materials required: Wash Buffer (0.1% sodium azide, 1% FBS in PBS); Fix Solution (1% FBS, 1 % formaldehyde in PBS); Fixable Viability Dye; FoxP3/Transcription Factor Fixation/Permeabilization Kit. All steps at room temperature, unless otherwise stated.

1. Pre-conjugate V5-tagged Ankyron™ in PBS with anti-V5 Fluorotag for Ankyron™, 8 μl/test. We would recommend trying an Ankyron™ titration range from 4 – 0.1 μg per test condition in the first instance. Allow 50 – 100 μL/sample. Shield from light and incubate at 4 °C for 30 min or overnight.

If also staining for cell surface proteins, follow steps 1 – 4 in the relevant section above, before permeabilizing cells ahead of detection of nuclear target proteins.

2. Apply Fixable Viability Dye in PBS to the cells, as per the manufacturer’s instructions, and mix well. Incubate for 15 min at +4 °C. Typically, 100 μL/well is sufficient. (Optional, but recommended). (Optional, but recommended).

3. Wash cells, resuspend in residual volume and fix samples using the FoxP3 Transcription Factor Staining Kit, as per the manufacturer’s instructions, for 45 min.

4. Wash cells in an excess of FoxP3 Permeabilization Buffer.

5. Add the pre-conjugated V5-tagged Ankyron™ with anti-V5 Fluorotag for Ankyron™ and any additional labelled Ankyrons™ or antibodies required for staining nuclear targets to the cells. Mix well and incubate samples at +4 °C for 30 minutes. The length and temperature of the incubation may require optimising, either at +4 °C or room temperature for 30 min or up to 16 h.

6. Wash cells with FoxP3 Permeabilization Buffer 2 – 3 times.

7. Resuspend cells in residual volume then add 200 μL/well Wash Buffer. Store samples in the dark at 4 °C until analysis.

Ankyrons™ in Fluorescence Microscopy

Centrifuge Ankyron™ in a chilled microcentrifuge at 14,000 xg for 5 min. This will remove protein aggregates that contribute to non-specific staining.

All steps at room temperature, unless otherwise stated.
Do not allow cells to dry out during the staining procedure.
The following protocols are suitable for staining cells in Chambered Cell Culture Slides: polystyrene chambers affixed to glass microscope slides, or 96-well Phenoplates.
After staining, ideally image immediately, or store samples at 4°C in the dark

Detection of extracellular target proteins in adherent cells

Additional materials required:
Wash Buffer (1x Phosphate Buffered Saline, PBS).
Blocking Buffer (10 % v/v Human Serum or Foetal Bovine Serum in RPMI 1640 medium)
If carrying out fixation: Fixative Buffer (1% formaldehyde1 in PBS)

Steps 1 -3 can be omitted if no fixation is required. Simply remove culture media from the cells and proceed with Step 4.

1. Washing. Remove culture media and wash cells with Wash Buffer.

2. Fixation. Apply Fixative Buffer and incubate for 5 min.

3. Washing. Remove Fixative Buffer, apply Wash Buffer then remove, proceeding to Step 4 immediately.

4. First Layer. Apply V5-tagged Ankyron™ in Blocking Buffer, typically 5 – 10 μg/ml, and incubate for 30 min.

5. Washing. Remove First Layer solution, apply Wash Buffer then remove, proceeding to Step 6 immediately.

6. Second Layer. Apply Ankyron™ Anti-V5 Fluorotag for Ankyron™ in Blocking Buffer, and incubate for 30 min. This can be omitted if the Ankyron™ is directly conjugated with a fluorophore.

7. Washing. Remove Second Layer solution, apply Wash Buffer then remove, proceeding to Step 8 immediately.

8. Nuclei Counterstain. DAPI (4′,6-Diamidino-2-Phenylindole) e.g. from BioLegend, use according to the manufacturer’s recommendations.

9. Fixation. Apply Fixative Buffer and, if carrying out staining and imaging in a 96-well Phenoplate, ideally image immediately or store samples at +4 °C in the dark until imaging can be carried out. If using chamber slides, incubate cells for 5 min then remove Fixative Buffer, applying PBS to the cells in the chambers for the duration of incubation in a solvent bath (typically 10 – 15 min, according to the manufacturer’s instructions) to dissolve the external glue securing the polystyrene chambers to the slide.

10. Mounting. (Only necessary if using chamber slides). Remove excess fluid from the slide and apply mounting medium e.g. VectaShield antifade and coverslip. Then apply sealant around the edges, e.g. Biotium CoverGrip Coverslip Sealant and image ideally immediately or store at +4 °C in the dark until imaging can be carried out.

Detection of intracellular (non-nuclear) target proteins in adherent cells

Additional materials required:
Wash Buffer (1x Phosphate Buffered Saline, PBS).
Blocking and Permeabilization Buffer (10 % v/v Human Serum or Foetal Bovine Serum + Saponin2 (from Quillaja bark, 0.5 % w/v, in RPMI 1640 medium)
If carrying out fixation: Fixative Buffer (1% formaldehyde1 in PBS)

Steps 1 -3 can be omitted if no fixation is required. Simply remove culture media from the cells and proceed to Step 4.
If the target protein is localised in cell-cell junctions, this permeabilization protocol may aid detection.

1. Washing. Remove culture media and wash cells with Wash Buffer.

2. Fixation. Apply Fixative Buffer and incubate for 5 min.

3. Washing. Remove Fixative Buffer, apply Wash Buffer then remove, proceeding to Step 4 immediately.

4. First Layer. Apply V5-tagged Ankyron™ in Permeabilization and Blocking Buffer, typically 5 – 10 μg/mL, and incubate for 30 min.

5. Washing. Remove First Layer solution, apply Wash Buffer then remove, proceeding to Step 6 immediately.

6. Second layer. Apply Ankyron™ Anti-V5 Fluorotag for Ankyron™ in Permeabilization and Blocking Buffer, and incubate for 30 min.

7. Washing. Remove Second Layer solution, apply Wash Buffer then remove, proceeding to Step 8 immediately.

8. Nuclei Counterstain. DAPI (4′,6-Diamidino-2-Phenylindole) e.g., from BioLegend, use according to the manufacturer’s recommendations.

9. Fixation. Apply Fixative Buffer and, if carrying out staining and imaging in a 96-well Phenoplate, ideally image immediately or store samples at +4 °C in the dark until imaging can be carried out. If using chamber slides, incubate cells for 5 min then remove Fixative Buffer, applying PBS to the cells in the chambers for the duration of incubation in a solvent bath (typically 10 – 15 min, according to the manufacturer’s instructions) to dissolve the external glue securing the polystyrene chambers to the slide.

10. Mounting. (Only necessary if using chamber slides). Remove excess fluid from the slide and apply mounting medium e.g. VectaShield antifade and coverslip. Then apply sealant around the edges, e.g. Biotium CoverGrip Coverslip Sealant and image ideally immediately or store at +4 °C in the dark until imaging can be carried out.

Detection of nuclear target proteins in adherent cells

Additional materials required:
Wash Buffer (1x Phosphate Buffered Saline, PBS).
Nuclear Permeabilization Buffer (0.5 % v/v Triton X-1003 in PBS)
Blocking and Permeabilization Buffer (10 % v/v Human Serum or Foetal Bovine Serum + Saponin2 (from Quillaja bark, 0.5 % w/v, in RPMI 1640 medium)
If carrying out fixation: Fixative Buffer (1% formaldehyde1 in PBS)

Steps 2-3 can be omitted if no fixation is required.

1. Washing. Remove culture media and wash cells with Wash Buffer.

2. Fixation. Apply Fixative Buffer and incubate for 5 min.

3. Washing. Remove Fixative Buffer, apply Wash Buffer then remove, proceeding to Step 4 immediately.

4. Nuclear permeabilization. Apply Nuclear Permeabilization Buffer and incubate for 10 min, then wash with PBS.

5. First Layer. Apply V5-tagged Ankyron™ in Permeabilization and Blocking Buffer, typically 5 – 10 μg/mL, and incubate for 30 min.

6. Washing. Remove First Layer solution, apply Wash Buffer then remove, proceeding to Step 7 immediately.

7. Second layer. Apply Ankyron™ Anti-V5 Fluorotag for Ankyron™ in Permeabilization and Blocking Buffer, and incubate for 30 min.

8. Washing. Remove Second Layer solution, apply Wash Buffer then remove, proceeding to Step 9 immediately.

9. Nuclei Counterstain. DAPI (4′,6-Diamidino-2-Phenylindole) e.g., from BioLegend, use according to the manufacturer’s recommendations.

10. Fixation. Apply Fixative Buffer and, if carrying out staining and imaging in a 96-well Phenoplate, ideally image immediately or store samples at +4 °C in the dark until imaging can be carried out. If using chamber slides, incubate cells for 5 min then remove Fixative Buffer, applying PBS to the cells in the chambers for the duration of incubation in a solvent bath (typically 10 – 15 min, according to the manufacturer’s instructions) to dissolve the external glue securing the polystyrene chambers to the slide.

11. Mounting. (Only necessary if using chamber slides). Remove excess fluid from the slide and apply mounting medium e.g. VectaShield antifade and coverslip. Then apply sealant around the edges, e.g. Biotium CoverGrip Coverslip Sealant and image ideally immediately or store at +4 °C in the dark until imaging can be carried out.

Notes
1. Paraformaldehyde, PFA can be purchased as a methanol-free 16 % w/v aqueous solution in individual ampoules. Dilute to required concentration in PBS and discard 1 week after opening and dilution. Alternatively, PFA can be made in-house from PFA solid, by dissolving in ultrapure water at 2 % w/v, heating to no more than 55°C and adding sodium hydroxide to aid solubility, then adding an equal volume of 2X PBS and adjusting pH to 7.4 using hydrochloric acid (diluted 1 in 10). Again, discard unused PFA after 1 week: over time, formaldehyde in solution will break down into formic acid and methanol, decreasing concentration of formaldehyde available to cross-link and fix tissues. Formic acid may destroy epitopes whilst methanol can disrupt membranes.
2. Saponin, a non-ionic detergent derived from the soapbark tree (Quillaja Saponaria), available from Sigma-Aldrich, to transiently permeabilize the plasma membrane. Once saponin is removed, the membrane will recover and therefore saponin must be included in all steps of the staining protocol.
3. Triton X-100, a non-ionic detergent. At high concentrations, it will lyse cells but at this concentration it irreversibly permeabilizes membranes, including the nuclear membrane. The concentration may need to be optimized according to the cell line and may be tested in the range of 0.1 – 0.5 % v/v.
Hazards
Paraformaldehyde/formaldehyde can irritate the skin, throat, lungs and eyes, and is a probable human carcinogen. Use a fume hood if weighing out solid paraformaldehyde. Combustible liquid, harmful if swallowed or in contact with skin, causes serious eye and skin irritation and may be a human carcinogen.
Saponin is an irritant.
Triton X-100 is toxic, irritant and aquatic hazard
Wear protective clothing, safety glasses and gloves when following these protocols.

Ankyrons™ in ELISA

Additional materials required: Diluent Buffer (1% BSA, 0.05% Tween-20 in PBS), Wash Buffer (0.1% Tween-20 in PBS).

1. Add the biotinylated antigen of interest to each well of a streptavidin coated 96-well microtiter plate and incubate for 1 hour at room temperature. Antigens of interest may be bound to a microtiter plate by other methods.
2. Precomplex the Ankyron™ (V5, His-tagged) specific to the antigen of interest with 1x final concentration Anti-V5 HRP Tag for 1 hour at room temperature* (22 °C). We recommend trying an Ankyron™ titration range from 50 – 0.05 ng to each well of the microtiter plate in the first instance. This incubation step to pre-complex the Ankyron™ with antibody increases the signal intensity and thus sensitivity of the assay, as well as reducing experimental time.
3. Wash wells of the microtiter plate with ELISA Wash Buffer.
4. Add the precomplexed Ankyron™ (V5, His-tagged) and Anti-V5 HRP mixture to wells of the microtiter plate.
5. Incubate at room temperature (22 °C) for 1 hour.
6. Wash wells of the microtiter plate with Wash Buffer.
7. Add an HRP compatible substrate to each well of the microtiter plate and measure signal.

*Precomplexing of the bivalent anti-V5 reagent with the V5 tagged Ankyron results in the multimerization of the Ankyron clone increasing the avidity of interaction between Ankyron and target protein.

Ankyrons™ in Immunohistochemistry

Automated staining using the Leica BOND RX

Dewaxing, antigen retrieval and Ankyron™ staining is carried out using the following protocol with BOND Polymer Refine Detection (Leica DS9800) and DAB (brown) chromogen . Retrieval conditions for individual Ankyron™ clones may require optimising, e.g. using Epitope Retrieval buffer ER1 in place of ER2, with different lengths of heating.

Step Reagents Time/Temperature
1. Dewax BOND Dewax Solution, 100% Alcohol, BOND Wash Solution Pre-programmed Leica BOND
2. Antigen Retrieval BOND Epitope Retrieval ER2 Solution 10 min., 100˚C | Protocol: HIER 10 min with ER2
3. Peroxide Block Refine Detection Kit Peroxide Block* 5 min
4. Wash BOND Wash Solution 3x 0:00 min.
5. Primary layer Ankyron applied at 10 μg/mL in BOND Wash Solution 60 min
6. Wash BOND Wash Solution 3x 0:00 min.
7. Post Primary Refine Detection Post Primary* 8 min
8. Wash BOND Wash Solution 3x 2:00 min.
9. Secondary layer Anti-HRP (1 in 500) in BOND Wash Solution 30 min
10. Wash BOND Wash Solution 3x 2:00 min.
11. Secondary Detection Refine Detection Kit Polymer* 8 min
12. Wash BOND Wash Solution 3x 2:00 min.
13. Wash Deionized Water 1x 0:00 min.
14a. Visualization Refine Detection Kit Mixed DAB Refine* 0.00 min
14b. Visualization Refine Detection Kit Mixed DAB Refine* 10 min
15. Wash Deionized Water 3x 0:00 min.
16. Counterstain Refine Detection Kit Hematoxylin* 5 min
17. Wash Deionized Water 0:00 min.
18. Wash Bond Wash Solution 0:00 min.
19. Wash Deionized Water 0:00 min.

*These reagents are included in BOND Polymer Refine Detection Kit (Catalog no. DS9800)
Dry slides at 37 °C, then apply hard set mounting media (e.g. ThermoScientific 1859351) and coverslips in a fume hood and allow to set.

Low throughput method

Additional materials required

Xylene; 100 % Ethanol; 50 % Ethanol; Wash Buffer (Tris-buffered saline with 0.05 % Tween-20™); Universal Blocker Blocking Buffer (Pierce); anti-V5 HRP; DAB Substrate (e.g. Metal Enhanced DAB Substrate, Pierce); Hematoxylin, Mounting Media; Coverslips
Use ultrapure water (18 MΩ) throughout.

All incubation steps to be carried out at room temperature, unless otherwise stated.

Dewaxing, rehydration and antigen retrieval

Place the slides in a rack and perform the following washes in a solvent-resistant container:

Solution Incubation time
Xylene 5 min
Xylene 5 min
Xylene 5 min
100 % Ethanol 5 min
100 % Ethanol 5 min
50 % Ethanol 5 min
50 % Ethanol 5 min
Water 5 min
Water 5 min

Do not allow sections to dry out, as this will cause non-specific binding of Ankyrons and high background staining.

Transfer slides to Antigen Retrieval Buffer at room temperature and then place in a preheated oven, 60 °C, incubating overnight.

Wash slides in water, prewarmed to 37 °C and proceed immediately to the next step. To prevent wastage of reagents, use a hydrophobic barrier pen to draw a circle around the tissue specimens on the slides.

Ankyron™-mediated detection of target proteins

  1. Quench endogenous peroxidase activity with peroxidase suppressor. This step may be omitted in tissue with low peroxidase present.
  2. Wash slides in Wash Buffer, 3 min x 2.
  3. Apply Universal Blocker Blocking Buffer, 30 min.
  4. Apply V5-tagged Ankyron™, 10 μg/ml in Universal Blocker Blocking Buffer, 60 min.
  5. Wash slides in Wash Buffer, 3 min x 2.
  6. Apply anti-V5 HRP at manufacturer’s recommended concentration, 30 min.
  7. Wash slides in Wash Buffer, 3 min x 2.
  8. Prepare DAB Substrate as per the manufacturer’s instructions, apply to slides and incubate until the desired staining is achieved (typically 5 – 15 min).
  9. Wash slides in Wash Buffer, 3 min x 2.
  10. Rinse with water and drain excess water from the specimen.
  11. Apply Hematoxylin nuclei counterstain to cover the entire tissue surface and incubate for 1 – 2 min.
  12. Drain off the Hematoxylin and wash slides several times with water
  13. Wash slide in Wash Buffer for 1 min
  14. Wash slide in water
  15. Mount slide with Mounting Media and coverslip.

Ankyrons™ in Western blotting

All steps to be carried out at room temperature, unless otherwise stated.

Additional materials required:

  1. 1x TBS (Tris Buffered Saline, 0.05 M Tris, 0.15 M NaCl, pH 7.6)
  2. Wash Buffer 1 (1x TBS with 0.1 % Tween 20)
  3. Wash Buffer 2 (1x concentrated TBS (0.05 M Tris, 0.5 M NaCl, pH 7.6 with 0.5 % Tween 20)
  4. Blocking Buffer (5 % w/v skimmed milk powder in 1x TBST)
  5. Incubation Buffer (5 % BSA in Wash buffer 2)
  6. SDS PAGE non-reducing buffer
  7. 1x MOPS buffer
  8. Pre-stained protein marker
  9. SDS PAGE gels
  10. Trans-Blot Turbo transfer machine, pure methanol and 1x Trans-Blot turbo transfer buffer (Bio-Rad)
  11. Blotting membrane and filter stacks: This protocol has been optimized for 0.2 µm PVDF membranes
  12. Anti-V5 HRP
  13. HRP substrate: This protocol has been optimized for colorimetric TMB substrate

Prepare samples for gel loading

  1. Combine the appropriate amount of sample, PBS and SDS PAGE non-reducing buffer.
  2. Briefly centrifuge the samples to move them to the bottom of the tube.
  3. Boil samples at 95 °C for 5 min.
  4. Briefly centrifuge samples to move all condensation to the bottom of the tube.

SDS PAGE and membrane transfer

  1. Run the samples on the appropriate SDS PAGE gel type in 1x MOPS buffer using pre-stained protein marker at 100 V for 90 min.
  2. When run has finished, assemble the blotting sandwich following the manufacturer’s instructions.
  3. Transfer using the Trans-Blot Turbo transfer machine for (2.5 A, 25V, 8 min).

Ankyron™-mediated detection of target proteins

  1. After transfer, wash membranes in 1x TBS for 5 min on a rocking platform.
  2. Incubate membranes in Blocking Buffer overnight at 4 °C.
  3. Pre-complex Ankyron™ (V5 tagged, 1ug/ml) and anti-V5-HRP (1:2000) in 5% BSA in 1x Wash Buffer 2 and mix on rocking platform for 1 h at room temperature.
    We recommend trying an Ankyron™ titration range from 1 – 0.2 ug/ml in the first instance. This incubation step to pre-complex the Ankyron™ with antibody increases the signal intensity and thus sensitivity of the assay, as well as reducing experimental time.
  4. Discard blocking buffer and briefly rinse membranes with MQ water
  5. Incubate membranes in pre-complexed Ankyron™ (V5 tagged) and Anti-V5 HRP for 1 h on a rocking platform.
  6. Discard the pre-complexed Ankyron™ (V5 tagged) and Anti-V5 HRP.
  7. Wash membranes once with Wash Buffer 2 for 5 min and then twice in Wash Buffer 1 for 2 x 5 min on rocking platform.
  8. Transfer the membranes onto a plastic tray and rinse briefly with MQ water to remove any remaining TBST.
  9. Add 0.5- 1 ml of colorimetric TMB substrate to each membrane and incubate for 5-15 min. Monitor the reaction and when protein bands are visible and background is still low, rinse membrane 2x in MQ water.
  10. Dry membranes to increase signal to background ratio.
  11. Perform detection of colorimetric signal.