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Western Blot: Technique, Theory, and Trouble Shooting

Western blotting is an important technique used in cell and molecular biology. Researchers can identify specific proteins from a complex mixture of proteins extracted from cells using Western Blot.

A mixture of proteins is separated based on molecular weight through gel electrophoresis. The gels are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labels antibodies specific to the protein of interest. The bound antibodies are then detected by developing the film.

The multi-tag positive loading control protein is used to demonstrate that your Western Blot protocol is efficient and correct and that the antibody recognizes the target protein which may not be present in the experimental samples. Loading such protein into your positive control lane results in a reliably detectable protein sample. It means all the steps of your Western blot functioned adequately, including gel electrophoresis, protein transfer to blotting membrane, membrane blocking and antibody labeling. It also gives you greater confidence that the results in the other lanes are real rather than artifactual.

We strongly recommend the use of a positive control protein when setting up a new experiment; this will give you immediate confidence in your Western Blot protocol.

When trying to detect low-abundance proteins, it is especially important to know that your Western blot is functioning as expected. If you detect your protein of interest in the control lane, then an absence of the protein in other lanes is probably directly related to its low abundance rather than a faulty step in the blotting protocol.


  • Demonstrate that your protocol is efficient and correct.
  • Perfect positive control protein for your Western blotting.
  • Save time and resources for all your protein research projects.
  • Give you confidence in your Western blot protocol.
  • Can be used for immuno-precipitation, affinity purification, Western blot and dot blot.

Order this positive loading control protein now: https://lifetein.com/peptide-product/multitag-protein-p-1.html

This paper provides the theoretical explanation of the Western Blotting procedure, troubleshooting tips for common problems: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/

Troubleshooting: (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/)

Even though the procedure for western blot is simple, there could be many unexpected results:
(1) Unusual or unexpected bands
(2) No bands
(3) Faint bands or weak signal
(4) High background on the blot
(5) Patchy or uneven spots on the blot

(1) Unusual or unexpected bands: These can be due to protease degradation, which produces bands at unexpected positions. It is advisable to use a fresh sample which had been kept on ice or alter the antibody. If the protein seems to be in too high of a position, then reheating the sample can help to break the quaternary protein structure. Similarly, blurry bands are often caused by high voltage or air bubbles present during transfer. In this case, it should be ensured that the gel is run at a lower voltage and that the transfer sandwich is prepared properly. Also, changing the running buffer can also help the problem. Nonflat bands can be the result of too fast of travel through the gel, due to low resistance. To fix this, the gel should be optimized to fit the sample. Finally, white (negative) bands on the film are due to too much protein or antibody.

(2) No bands: This is due to many reasons related to the antibody, antigen, or buffer used. If an improper antibody is used, either primary or secondary, the band will not show. Also, the concentration of the antibody should be appropriate as well; if the concentration is too low, the signal may not be visible. It is important to remember that some antibodies are not to be used for western blot. Another reason for no visible bands is the lowest concentration or absence of the antigen. In this case, an antigen from another source can be used to confirm whether the problem lies with the sample or with other elements, such as the antibody. Moreover, prolonged washing can also decrease the signal. Buffers can also contribute to the problem. It should be ensured that buffers like the transfer buffer, TBST, running buffer and ECL are all new and noncontaminated. If the buffers are contaminated with sodium azide, it can inactivate HRP.

(3) Faint bands or weak signal: It can be caused by low concentration of antibody or antigen. Increasing exposure time can also help to make the band clearer. Another reason could be nonfat dry milk masking the antigen. In this case, use BSA or decrease the amount of milk used.

(4) High background on the blot: It is often caused by the too high concentration of the antibody, which can bind to PVDF membranes. Another problem could be the buffers, which may be too old. Increasing the washing time can also help to decrease the background. Additionally, too high of exposure can also lead to this problem. Therefore, it is advisable to check different exposure times to achieve an optimum time.

(5) Patchy or uneven spots on the blot: Improper transfer usually causes them. If there are air bubbles trapped between the gel and the membrane, it will appear darker on the film. It is also essential to use a shaker for all incubation so that there is no uneven agitation during the incubation. Once again, washing is of utmost importance as well to wash the background. Antibodies binding to the blocking agents can also cause this problem; in this case, another blocking agent should be tried. Filtering the blocking agent can also help to remove some contaminants. Finally, this problem can also be caused by aggregation of the secondary antibody; in this case, the secondary antibody should be centrifuged and filtered to remove the aggregated.

ELISA standard curve
Standards and the standard curve 1. Make up a stock solution of 0.08 ug of prot...
LifeTein Dot Blot protocol

Dot Blot protocol

Dot blot is similar to the Western blot technique. However the proteins are spotted directly onto the membrane or paper for detecting and analyzing. This is a good technique to estimate the protein concentration.


1. Prepare the nitrocellulose (NC) membrane.

2. Spot 2 µl of diluted samples onto the NC membrane and let it dry.

3.Blocking: Block the membrane in 1% BSA or non-fat milk in TBST (1 hr, Room Temperature).

4. Primary Antibody: Incubate with primary antibody in BSA/TBST for 1 hr at RT.

5. Washing: Wash three times with TBST (3 x 5 min).

6.Secondary antibody: Incubate with secondary antibody conjugated with HRP for 1 hr at RT.

7. Washing: Wash three times with TBST (15 min x 1, 5 min x 2), then once with TBS (5 min).

8. Developing: Incubate with ECL reagent for 1 min and expose X-ray film in the dark room with different lengths of exposure.



20 mM Tris-HCl

150 mM NaCl

pH 7.5


0.05% Tween20 in TBS


0.1% BSA in TBST

NC: Nitrocellulose membrane (BIO-RAD)

LifeTein ELISA Protocol

ELISA Protocol

Coating the appropriate antigen to microplate

1. Dilute the antigen to a final concentration of 20 μg/ml in PBS. Fill the microwells of a

Nunc Maxi-Sorp Immuno Plate with 50 μL of the diluted antigen.

Note: Test samples containing pure antigen are usually pipeted onto the plate at less

than 2 μg/ml. Antigen protein concentration should not be over 20 μg/ml as this will

saturate most of the available sites on the microtitre plate.

2. Incubate at 4 °C overnight or 2 h at room temperature.

3. Wash the unbound antigen off the plate by flicking the contents of the plate into the

sink, fill the wells with DI water, flick again, repeat 2X with PBS-Triton.


4. Block the remaining protein-binding sites in the coated wells by adding 200 μl

blocking buffer, 1% BSA/PBS or other blocking reagents.

5. Incubate for 30-60 minutes at Room Temperature (RT) or 4 °C overnight.

6. Wash plate as above.

Incubation with the antibody

7. Add 100 μl of the antibody, diluted at the optimal concentration in blocking buffer

immediately before use.

Note: Be sure to include positive and negative controls, and, if necessary, a standard


8. Incubate for 2 h at room temperature.

Note: 2 hours is usually enough to obtain a strong signal. Stronger staining will often

observed when incubated overnight at 4 °C.

9. Wash the plate 4 times with PBS.


10. Dispense 100 μl (or 50 μl) of the substrate solution per well with a multichannel


11. After sufficient color development add 100 μl of stop solution to the wells.

12. Read the absorbance (optical density) of each well with an ELISA plate reader.

Common Substrates and the appropriate plate reader setting

ABTS: 405-410 nm

TMB: non-stopped 620-650 nm, stopped 450 nm

OPD: non-stopped 450 nm, stopped 490 nm

pNPP: 405-410 nm

BluePhosTM: 595-650 nm

Buffers and reagents

Bicarbonate/carbonate coating buffer (100 mM)

3.03 g Na2CO3,

6.0 g NaHCO3

1000 ml distilled water

pH 9.6,


1.16 g Na2HPO4,

0.1 g KCl,

0.1 g K3PO4,

4.0 g NaCl (500 ml distilled water) pH 7.4.

Blocking solution

1% BSA, serum, non-fat dry milk, casein, gelatin in PBS.

Wash solution

PBS or Tris-buffered saline (pH 7.4) with 0.05% (v/v) Tween20 (TBST) or Triton.

Antibody dilution buffer

Primary and secondary antibody should be diluted in 1x blocking solution to reduce nonspecific


Antibody Storage
**Guidelines for the storage of different types of antibody** Storage temperat...