C. Transfer of proteins and staining (WB guide)

Transfer of proteins and staining protocols. Section C from our extensive beginners' guide to western blot

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Contents

  1. Visualize of proteins in gels
  2. Transfer
  3. Visualization of proteins in membranes: Ponceau Red
  4. Blocking the membrame
  5. Incubation with the primary antibody
  6. Incubation with secondary antibody
  7. Development methods

1. Visualization of proteins in gels

This visualization of protein at this stage is useful to determine if proteins have migrated uniformly and evenly. Use the copper stain if you plan to transfer the separated proteins to a membrane, as the Coomassie stain is not reversible. Only use the Coomasie stain on gels post-transfer to check the efficiency of the transfer, or if you have no plans to transfer and just want to observe the results of the SDS-PAGE separation.

a) Coomassie stain
As soon as the power is turned off the separated protein bands will begin to diffuse (they are freely soluble in aqueous solution). To prevent diffusion of proteins treat the gel with a 40% distilled water, 10% acetic acid, and 50% methanol solution which causes almost all proteins to precipitate (become insoluble). To visualize the fixed proteins place the gel in the same mixture of water/acetic acid/methanol but with the addition of 0.25% by weight Coomassie Brilliant Blue R-250. Incubate 4 hours to overnight at room temperature on a shaker. Transfer the gel (save the dye mixure; it can be re-used many times) to a mixture of 67.5% distilled water, 7.5% acetic acid, and 25% methanol, place on shaker, and replace with fresh rinse mixture until the excess dye has been removed. The stain will not bind to the acrylamide, and will wash out (leaving a clear gel). However, it remains strongly bound to the proteins in the gel, and these take on a deep blue color.

b) Copper stain
Briefly rinse freshly-electrophoresed gels in distilled water (30 seconds maximum) and then transfer to 0.3 M CuCl2 for 5 to 15 minutes. Wash the gels briefly in de-ionized water, and view them against a dark-field background. Proteins come up as clear zones in a translucent blue background. Gels may be destained completely by repeated washing in 0.1- 0.25 M Tris/0.25 M EDTA pH 8.0. Move the gel to a dish of transfer buffer before proceeding with transfer according to the transfer apparatus manufacturer’s instructions.

2. Transfer

Detailed instructions for the transfer process can be found on the websites of the manufacturers of transfer apparatus, and will vary depending on the system. The principle is the same in each case, though. Just as proteins with an electrical charge (provided by the SDS bound to them) can be induced to travel through a gel in an electrical field, so can the proteins be transferred in an electrical field from the gel onto a sturdy support, a membrane that “blots” the proteins from the gel. (Early methods relied on diffusion; blotting in an electrical field is now standard).

Transfer can be done in wet or semi-dry conditions. Semi-dry transfer is generally faster but wet transfer is a less prone to failure due to drying of the membrane and is especially recommended for large proteins, >100 kD. For both kinds of transfer, the membrane is placed next to the gel. The two are sandwiched between absorbent materials, and the sandwich is clamped between solid supports to maintain tight contact between the gel and membrane.

In wet transfer, the gel and membrane are sandwiched between sponge and paper (sponge/paper/gel/membrane/paper/sponge) and all are clamped tightly together after ensuring no air bubbles have formed between the gel and membrane. The sandwich is submerged in transfer buffer to which an electrical field is applied. The negatively-charged proteins travel towards the positively-charged electrode, but the membrane stops them, binds them, and prevents them from continuing on.

A standard buffer for wet transfer is the same as the 1X Tris-glycine buffer used for the migration/running buffer without SDS but with the addition of methanol to a final concentration of 20%. For proteins larger than 80 kD, it is recommended that SDS is included at a final concentration of 0.1%.

In semi-dry transfer, a sandwich of paper/gel/membrane/paper wetted in transfer buffer is placed directly between positive and negative electrodes (cathode and anode respectively). As for wet transfer, it is important that the membrane is closest to the positive electrode and the gel closest to the negative electrode. The proportion of Tris and glycine in the transfer buffer is not necessarily the same as for wet transfer; consult the apparatus manufacturer’s protocol. A standard recipe is 48 mM Tris, 39 mM glycine, 0.04% SDS, 20% methanol.

Two types of membranes are available: nitrocellulose and PVDF (positively-charged nylon). The choice is personal and both work very well. PVDF membranes require careful pre-treatment: cut the membrane to the appropriate size then soak it in methanol for 1-2 min. Incubate in ice cold transfer buffer for 5 minutes. The gel needs to equilibrate for 3-5 minutes in ice cold transfer buffer. Failure to do so will cause shrinking while transferring, and a distorted pattern of transfer.

Note on transfer of large and small proteins

The balance of SDS and methanol in the transfer buffer, protein size, and gel percentage can affect transfer efficiency. The following modifications will encourage efficient transfer.

Large proteins (>100 kD)

  1. For large proteins, transfer out of the gel may be very slow, just as they run slowly within the gel during separation. If blotting a large protein, be sure to run your samples in a low-concentration gel, 8% or less. These will be very fragile, so handle carefully.
  2. Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation.
  3. Lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily.
  4. Methanol is only necessary if using nitrocellulose. If using PVDF, methanol can be removed from the transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich.
  5. Choose wet transfer overnight at 4°C instead of semi-dry transfer.

Small proteins (<100 kD)

  1. All proteins are hindered from binding to membranes by SDS but small proteins more so than large proteins. If your protein of interest is small, consider removing SDS from the transfer buffer.
  2. Keep the methanol concentration at 20%.

The following reference discusses a gel and buffer system that allows transfer of proteins as large as 500 kD:

Bolt and Mahoney, High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate–polyacrylamide, gel electrophoresis. Analytical Biochemistry 247, 185–192 (1997).

More transfer tips:

Top tip Molly

Avoid touching the membrane with your fingers; use tweezers instead. Oils and proteins on the fingers will block efficient transfer and create dirty blots.

After sandwiching the gel and membrane between paper, air bubbles between the gel and membrane can be removed by rolling them out with a pipet or 15 ml tube, or by assembling the sandwich in a dish of transfer buffer to prevent formation of bubbles in the first place. Wear gloves!

Make sure the paper and membrane are cut to the same size as the gel. Large overhangs may prevent a current from passing through the membrane in semi-dry transfers.

Chicken antibodies tend to bind PVDF and other nylon-based membranes, leading to high background. Switching to a nitrocellulose membrane should help reduce background staining.

3. Visualization of proteins in membranes: Ponceau Red

To check for success of transfer, wash the membrane in TBST (for a TBST recipe, see below). Dilute the stock Ponceau Red 1:10. The stock is made of 2% Ponceau S in 30% trichloroacetic acid and 30% sulfosalicylic acid.
Incubate on an agitator for 5 min.
Wash extensively in water until the water is clear and the protein bands are well-defined.
The membrane may be destained completely by repeated washing in TBST or water. When using a PVDF membrane, re-activate the membrane with methanol then wash again in TBST.

TBS 10x (concentrated TBS)
24.23 g Trizma HCl/
80.06 g NaCl
Mix in 800 ml ultra pure water.
pH to 7.6 with pure HCl.
Top up to 1 L.

TBST
For 1 L: 100 ml of TBS 10x + 900 ml ultra pure water + 1ml Tween20

Tween20 is very viscous and will stick to the tip of your measuring pipettes. Be sure you add the right amount of the detergent to the Tris buffer. A 10% solution is easier to dispense than undiluted Tween20.

4. Blocking the membrane

Blocking the membrane prevents non-specific background binding of the primary and/or secondary antibodies to the membrane (which has a high capacity at binding proteins and therefore antibodies).

Two blocking solutions are traditionally used: non-fat milk or BSA (Cohn fraction V). Milk is cheaper but is not recommended for studies of phospho-proteins (milk contains casein which is a phospho-protein; it causes high background because the phospho-specific antibody detects the casein present in the milk).

Some antibodies give a stronger signal on membranes blocked with BSA as opposed to milk for unknown reasons. Check the application notes on the datasheet in case there are specific instructions on how to block the membrane.

To prepare a 5% milk or BSA solution, weigh 5 g per 100 ml of Tris Buffer Saline Tween20 (TBST) buffer. Mix well and filter. Failure to filter can lead to “spotting” where tiny dark grains will contaminate the blot during development.

Incubate for 1 hour at 4°C under agitation. Rinse for 5 seconds in TBST after the incubation.

5. Incubation with the primary antibody

Incubation buffer: Dilute the antibody in TBST at the suggested dilution, if the datasheet does not have a recommended dilution try a range of dilutions (1:100-1:3000) and optimize the dilution according to the results. Too much antibody will result in non-specific bands.

It is traditional in certain laboratories to incubate the antibody in blocking buffer, while other laboratories incubate the antibody in TBST without a blocking agent. The results are variable from antibody to antibody and you may find it makes a difference to either use no blocking agent in the antibody buffer or the same agent as the blocking buffer.

Top tip Molly

If high background is not an issue, some antibodies produce a much stronger signal if diluted in buffer with low concentrations (0.5 – 0.25%) of milk or BSA, or none at all.

Incubation time: The time can vary between a few hours and overnight (rarely more than 18 hours), and is dependent on the binding affinity of the antibody for the protein and the abundance of protein. We recommend a more dilute antibody and a prolonged incubation to ensure specific binding.

Incubation temperature: preferably cold. If incubating in blocking buffer overnight, it is imperative to incubate at 4°C or contamination will incur and thus destruction of the protein (especially phospho groups).
Agitation of the antibody is recommended to enable adequate homogenous covering of the membrane and prevent uneven binding.

6. Incubation with secondary antibody

Wash the membrane several times in TBST while agitating, 5 minutes or more per wash, to remove residual primary antibody.

Incubation buffer and dilution: Dilute the antibody in TBST at the suggested dilution. If the datasheet does not have a recommended dilution, try a range of dilutions (1:1000- 1:20,000) and optimize the dilution according to the results. Too much antibody will result in non-specific bands. You may incubate the secondary antibody (and primary antibody) in blocking buffer, but a reduction in background may come at the cost of a weaker specific signal, presumably because the blocking protein hinders binding of the antibody to the target protein.

Incubation time and temperature: 1-2 hours, room temperature, with agitation.

Which conjugate?We recommend HRP-conjugated secondary antibodies. ALP-conjugated secondary antibodies (alkaline phosphatase) are not recommended as they are not sensitive enough.

7. Development methods

Detection kits
For HRP-conjugated antibodies: ECL and ECL+ (home made or commercially available) are the traditional kits used and we recommend ECL+. For the new generation detection machines such as Genegnome, use the detection kit recommended by the manufacturer of the machine.

We do not recommend ECL or BCIP/NBT detection kits as they are not as sensitive.

X-ray films
Manual film development is traditionally used and enables the scientist to control the incubation time of the x-ray film in the developing agent and fixation agent.
Automated x-ray film developers are also widely used and easy to use.

Top tip Molly

Remember that an over-exposed film is not suitable for analysis as determination of the relative amount of protein is not possible. Overexposed films show totally black bands with no contrast, and/or numerous non-specific bands.

Digital images:
The new generation of film developers are units with a camera inside an enclosure, removing the need for a darkroom. The camera detects the chemiluminescence emanating from the membrane, transforming the signal into a digital image for rapid analysis with software provided with the detection machine.

A range of machines are now commercially available. At the front of the next generation are systems which do not use HRP-conjugated antibodies (i.e chemiluminescence): for example, STORM Analysers detect fluorescence from fluorochrome-conjugated secondary antibodies. The Odyssey Infrared Imaging System detects infrared fluorescence.

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