For nitrocellulose or PVDF membrane following Western blot detection using a chemiluminescent or fluorescent substrate system: Following transfer, air dry the membrane and place in an envelope, preferably on top of a supported surface to keep the membrane flat. The blot can be stored indefinitely at -80 degrees C. When ready to reprobe, prewet the PVDF blot with alcohol for a few seconds, followed by a few rinses with pure water to reduce the alcohol concentration. Then proceed as normal with blocking step.

FOR STRIPPING/REPROBING OF MEMBRANES: Harsh protocol (see NOTE below for modifications)

1) Submerge the membrane in stripping buffer (100 mM BME, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubate at 50 degrees C for 30 min with occasional agitation. If more stringent conditions necessary, incubate at 70 degrees C.

2) Wash 2 x 10 min in TBS-T/PBS-T at room temperature.

3) Block the membrane by immersing in 5% blocking reagent TBS-T or PBS-T for 1 hr at room temperature.

4) Immunodetection

NOTE: Often you don't need such harsh conditions to remove antibodies from their proteins. The stringency of one or several of the variables can be decreased: lower the temperature, decrease the time, less BME, less SDS, etc. An especially mild but still often effective stripping protocol is lower pH incubation. Example: pH 2.0 Tris 50-100 mM, 30-60 min incubation (you may do two incubations if you wish). Then rinse and block as usual. If you do not wish to re-use the membrane immediately after stripping, you can store the membrane in plastic wrap (wet, you do not want it to dry out). Another simple, mild stripping buffer is 0.1 M glycine•HCl (pH 2.5-3.0), incubation 30 min to 2 hrs room temperature or 37 degrees C, depending on the antibody.

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For IEF gels, we recommend using an acetic acid transfer buffer. The IEF gels are 5% polyacrylamide and transferring them in a basic buffer leads to substantial hydrolysis and damage to the gel. The following protocol prevents the gel from hydrolyzing and is especially effective for basic proteins because of the low pH of the transfer buffer:

1) After the run, equilibrate the gel in 0.7% acetic acid (0.7% acetic acid in water, pH 3.0) for 10 min.

2) Chill the 0.7% acetic acid that will be used as the transfer buffer.

3) Assemble the gel/membrane sandwich in reverse order so that the membrane is in contact with the side of the gel facing towards the cathode (-). This is OPPOSITE from the typical western blot with SDS-PAGE, where the negatively-charged protein will migrate toward the anode (+) side during the transfer.

4) Transfer for one hour at 10 V.

Tip for handling the IEF gel: the 5% polyacrylamide IEF gels tend to be sticky. While the gel is floating in the equilibration solution, submerge the filter paper underneath the gel. When the gel is in the correct position, lift up on the filter paper so the gel attaches to it. Floating the gel over the filter paper avoids the need to handle the gel and prevents the gel from getting stuck onto the filter paper before it is in its proper position.

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For IEF gels, we recommend using an acetic acid transfer buffer. The IEF gels are 5% polyacrylamide and transferring them in a basic buffer leads to substantial hydrolysis and damage to the gel. The following protocol prevents the gel from hydrolyzing and is especially effective for basic proteins because of the low pH of the transfer buffer:

After the run, equilibrate the gel in 0.7% acetic acid (0.7% acetic acid in water, pH 3.0) for 10 minutes. Chill the 0.7% acetic acid that will be used as the transfer buffer. Assemble the gel/membrane sandwich in reverse order so that the membrane is in contact with the side of the gel facing towards the cathode (-). This is opposite from the typical western blot with SDS, where the negatively charged protein will migrate toward the anode (+) side during the transfer. Transfer for one hour at 10 volts.

Tip for handling the IEF gel: The 5% polyacrylamide IEF gels tend to be sticky. While the gel is floating in the equilibration solution, submerge the filter paper underneath the gel. When the gel is in the correct position, lift up on the filter paper so the gel attaches to it. Floating the gel over the filter paper avoids the need to handle the gel and prevents the gel from getting stuck onto the filter paper before it is in its proper position.

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For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternDot immunodetection protocol.

Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternDot immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternDot immunodetection protocol.

For Native-PAGE Western blots, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 minutes before proceeding to the WesternDot immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternDot immunodetection protocol.

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For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternBreeze Chromogenic Immunodetection protocol.

Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternBreeze Chromogenic Immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 mins before proceeding to the WesternBreeze Chromogenic Immunodetection protocol.

For Native-PAGE western blot, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 mins before proceeding to the WesternBreeze Chromogenic Immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternBreeze Chromogenic Immunodetection protocol.

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For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternBreeze Chemiluminescent Immunodetection protocol.

Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternBreeze Chemiluminescent Immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 mins before proceeding to the WesternBreeze Chemiluminescent Immunodetection protocol.

For Native-PAGE western blot, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 mins before proceeding to the WesternBreeze Chemiluminescent Immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternBreeze Chemiluminescent Immunodetection protocol.

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Optimize the concentration of primary and secondary antibodies. In some cases, increasing the concentration of blocking agent (BSA or non-fat dry milk) or usiing an alternative blocking solution such as Starting Block or SuperBlock may reduce background signal. After incubation with the primary antibody, wash at least 2 times with TBST (include 0.5 M NaCl in one or more of the wash steps). Avoid Nonidet P40 or Triton X-100 in buffers because protein detection is decreased when these detergents are used.

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PVDF membranes require more stringent blocking steps. This can be achieved by increasing the concentration of the blocking reagent 2 to 5 fold, increasing the blocking time, and performing the procedure at 37 degrees C. Blocking agents bind to unoccupied sites to prevent background staining and also to membrane-bound proteins, thus reducing non-specific interactions with the primary antibody. We offer WesternBreeze Immunodetection kits with blocking reagents and Pierce Fast Western Blot kits that have been pre-optimized to give low-background blots. Other examples of blocking agents are nonfat dry milk, BSA, and casein, Starting Block, and SuperBlock.

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No, we do not recommend staining membranes with the Colloidal Blue (G-250) Staining Kit as the background will be too high. Better alternatives include:

- Coomassie (non-colloidal) staining: Stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 minutes and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at -20 degrees C. Sensitivity is approximately at the 50-100 ng level.
- SimplyBlue SafeStain. There is a protocol included in the SimplyBlue SafeStain manual for staining PVDF but it is not recommended for nitrocellulose because of the high background.
- Amido Black: same as Coomassie but less sensitive.
- Ponceau S: same as Coomassie but less sensitive.
- UV transillumination: After blotting, place the membrane on a filter paper and allow to dry at room temperature for about 10 minutes. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear because the membrane is dry, rewet the membrane.
- Invitrogen Reversible Membrane Protein Stain (Cat# IB7710): Allows for complete, reversible staining of protein on nitrocellulose & PVDF membranes. Sensitivity is higher than Ponceau S (less than 10 ng of BSA in 10 mins as blue bands) and the staining is reversible in 5 minutes. Western blot detection is unaffected by the staining and erasing process, and in some cases higher sensitivity is achieved.

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No we do not recommend staining membranes with the Colloidal Blue (G-250) Staining Kit as the background will be too high. Better alternatives include:

- Invitrogen Reversible Membrane Protein Stain (Cat. No. IB7710): Allows for complete, reversible staining of protein on nitrocellulose & PVDF membranes. Sensitivity is higher than Ponceau S (<10 ng of BSA in 10 mins as blue bands) and the staining is reversible in 5 minutes. Western blot detection is unaffected by the staining and erasing process, and in some cases higher sensitivity is achieved.
- Coomassie (non-colloidal) staining: Stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 minutes and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at −20 degrees C. Sensitivity is approximately at the 50-100 ng level.
- SimplyBlue SafeStain. There is a protocol included in the SimplyBlue SafeStain manual for staining dry PVDF membranes but it is not recommended for wet PVDF membranes or nitrocellulose because of the high background.
- Amido black: same as Coomassie stain but less sensitive. Please see protocol in the manual (https://tools.thermofisher.com/content/sfs/manuals/invitrolonpvdf_man.pdf).
- Ponceau S: same as Coomassie stain but less sensitive. Please see protocol in the manual (https://tools.thermofisher.com/content/sfs/manuals/invitrolonpvdf_man.pdf).
- UV transillumination: After blotting, place the membrane on a filter paper and allow to air-dry at room temperature for about 10 minutes. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear because the membrane is dry, rewet the membrane.

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Consider transferring to a different membrane or using a different detection method. We have observed increased sensitivity when using PVDF membranes in place of nitrocellulose. On PVDF membranes, using as little as 1 µg of total rat brain protein, PKC can be detected with alkaline phosphatase-mediated chromogenic detection in some cases using affinity-purified antibodies at a concentration of 0.5 µg/mL. Detection sensitivity can also be increased by using chemiluminescent detection, especially when using a SuperSignal West enhanced chemiluminescence subtrate (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/western-blot-detection-reagents/detection-technologies-western-blotting/chemiluminescent-western-blot-detection/supersignal-chemiluminescent-substrates.html) such as SuperSignal West Pico PLUS, SuperSignal West Dura, or SuperSignal West Femto. The secondary antibody should be used as recommend by the manufacturer and optimized as needed.

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It is not recommended because the background will be too high. Better alternatives include: 1) Invitrogen Reversible Membrane Protein Stain Kit (Cat. No. IB7710).

2) Coomassie (non-colloidal) staining: stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 min and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at -20°C. Sensitivity is approximately at the 50-100 ng level.

3) Use SimplyBlue SafeStain (Cat. No. LC6060). The SimplyBlue SafeStain manual has the protocol for staining PVDF membranes, but it is not recommended for nitrocellulose because of high background.

4) Amido Black: same as Coomassie but less sensitive.

5) Ponceau S: same as Coomassie but less sensitive.

6) UV transillumination: place membrane on filter paper after blot is finished and allow to dry at room temperature for about 10 min. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear as the membranes dries, rewet again.

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PVDF membranes require more stringent blocking steps. This can be achieved by increasing the concentration of the blocking agent 2-5 fold, increasing the blocking time, and performing the procedure at 37 degrees C. Blocking agents bind to unoccupied sites to prevent background staining and also to membrane-bound proteins, reducing non-specific interactions with the primary antibody. Examples of blocking agents are nonfat dry milk, BSA, and Casein.

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SDS:
-Mobility out of the gel: increases mobility as SDS imparts negative charge to a protein and maintains it in a soluble state.
-Binding to membrane: reduces binding to nitrocellulose due to decreased hydrophobicity of the protein.
-Detection: may affect antigenicity of some proteins.

Alcohol in transfer buffer (i.e., methanol up to 20%):
-Mobility out of the gel: decreased; reduces pore size of gel.
-Binding to membrane: improves binding to nitrocellulose; removes SDS from proteins resulting in improved hydrophobic interactions.

Field strength (V/cm):
-Mobility out of the gel: field strength is the driving force of elution; if too low, sample is not completely transferred out of gel.
-Binding to membrane: if too high, sample passes through membrane without binding.

Transfer buffer:
-Mobility out of the gel: decreased if high conductivity and low resistance limit V/cm due to excessive heat generation; decreased if buffer pH is close to pI of native protein.

Membrane:
-Detection: PVDF and nylon require more stringent blocking conditions than nitrocellulose.

Gel:
-Mobility out of the gel: increases with thinner gels or larger gel pore size.

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