These are two very different methods althought they are both based on immunodetection. The advandages and disadvantages of these methods largely depends on your purpose. ELISA is to choose if you want to quantify a specific protein which is in a mix of various proteins, for instance in cell lysates or serum. If you want to see the size of your protein you should use WB. WB is to some degree also used to quantify but is not nearly as reliable as ELISA for that purpose, while in ELISA you will not see any size changes to your protein as long as the epitope of the protein that is recognized in the ELISA is there. Also, if you are screening many samples for the concentration of a specific protein, ELISA is definitely to prefere.

These are two very different methods althought they are both based on immunodetection. The advandages and disadvantages of these methods largely depends on your purpose. ELISA is to choose if you want to quantify a specific protein which is in a mix of various proteins, for instance in cell lysates or serum. If you want to see the size of your protein you should use WB. WB is to some degree also used to quantify but is not nearly as reliable as ELISA for that purpose, while in ELISA you will not see any size changes to your protein as long as the epitope of the protein that is recognized in the ELISA is there. Also, if you are screening many samples for the concentration of a specific protein, ELISA is definitely to prefere.

There is almost nothing to add to Alenka's answer, except one small thing: WBs are also used to verify the presence of one specific enzyme, e.g. during an overexpression procedure, either by epitope tags or by specific polyclonal/monoclonal target antibodies.

Western Blots are typically done to determine the purity and MW of a protein. There are done to determine if a specific protein is in the sample. One does this by adding an antibody that is specific for the protein on interest. The antibody binds to the protein and can be detected various ways.

Western blotting is effective and useful method to detect and characterize proteins in small amounts, such as clock proteins. Moreover, clock proteins’ other properties like half-life, molar amounts can also be found using western blotting.

Advantages

•Immunogenic responses from infectious agents (ex. viruses, bacteria) are hard to find since they are difficult to isolate from patient sample. But Western blotting can detect this.

•Western Blotting utilizes not only antigens, but also antisera as a diagnostic tool. Antisera is widely used in the test for HIV presence.

•Compared to ELISA, Western blotting has higher specificity; the higher specificity, the more the method is independent of the specificity of antibodies.

•Polyvinylidene difluoride (PVDF), or Nylon, is often used as membrane in Western blotting, since it has a high protein-binding capacity and chemical stability. Even, some protein groups only bind to Nylon or favor strongly to it.

•Among three common enzyme substrates, Fluorescent and Chemiluminescent create light detectable through X-ray or scanners. This ability enables high levels of sensitivity and quicker processing time.

Disadvantages

A non-intended protein has a slight chance of reacting with the secondary anti-body, resulting in the labeling of an incorrect protein.

•Incidental phosphorylation or oxidation of proteins may result in multiple bands appearing after sample is processed.

•The appearance of bubbles may occur when transferring the sample from the gel/membrane sandwich and may also occur when incubating the sample with antibodies, resulting in a skewed band reading.

•If the transfer time is not sufficient when transferring proteins to the membrane, the larger proteins of higher molecular weight will not transfer properly, resulting in an incorrect or no band reading at all.

•Too much methanol in the transfer buffer decreases the transfer efficiency of proteins from the gel to the membrane; however methanol aids in protein binding to several different membranes, so a correct balance is required.

•Western Blotting is a very delicate process requiring the correct amounts of each component in order for successful identification of the presence of proteins. An imbalance in any step of the procedure may skew the entire process.

ELISA,Enzyme-linked immunosorbent assay, is usually done to detect the presence of an antibody or an antigen in a sample. In ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody. Since the labeled secondary antibody is directed against all antibodies of a given species (e.g. anti-mouse), it can be used with a wide variety of primary antibodies (e.g. all mouse monoclonal antibodies). The use of secondary antibody also provides an additional step for signal amplification, increasing the overall sensitivity of the assay.

http://answers.yahoo.com/question/index?qid=20091114180328AAICwPZ

ELISA = quantitative assessment with a well characterized antibody. A very stringent validation process is needed before one jumps into an ELISA.

WB = qualitative assessment amenable to "dirty" antibody mixtures ie., antiserums, etc. The first thing you do after making an antibody.

Both require good controls. The literature is full of meaningless blots and ELISAs due to improper controls.

Elisa = Quantitative and WB = Qualitative

I agree to Alenka's answer. ELISA and WB are different methods althought they are both based on immunodetection. The advandages and disadvantages of these methods largely depends on your purpose. If you only want to know whether a specific protein exists in a mix of various proteins, they both worked. ELISA is suitble for quantifying a specific protein which is in a mix of various proteins. but If you want to see the size of your protein you should use WB. WB is to some degree also used to quantify but is not nearly as reliable as ELISA for that purpose, while in ELISA you will not see any size changes to your protein as long as the epitope of the protein that is recognized in the ELISA is there. Also, if you are screening many samples for the concentration of a specific protein, ELISA is definitely to prefere.

I agree with all the above answers, that yes they are both different techniques which are dependent on immunodetection and should be viewed and applied according to the information you want to gain.

WB physically show the protein in a gel and can give the observer a good measure of the size and molecular weight of the detectable protein. depending on the antibodies and controls used, this method can be used not only for qualitative determination of protein quantity but it can also be used to view degradation, digestion and multimerisation on the protein. You can only run as many WB simultaneously as the number of apparati you have and therefore the number of samples is limited.

ELISA is a powerful technique used to quickly determine either the presence (i.e. screening) or the amount (used in correlation with standard curves) of a particular protein. This method however doesn't take into account that many proteins form multimers (di- tri- tetra- penta- etc. mers) with itself or other isoforms of the protein. since ELISAs are performed in plates and require only incubation rather than electrophoresis, you can run as many assays as you want.

I hope this outlines the main advantages and disadvantages of these techniques and gives you an idea of which (or both) you should consider performing to gain the information you require about your protein :)

In general, ELISA is more sensitive than WB and WB is more specific than ELISA.

ELISA can measure minimum amount of protein

1) I do agree with Meysam.

2) As far as I know ELISA is only for secreted proteins whereas WB can be done for both secreted and intracellular proteins.

@ Agnieszka Pastula : are you sure about this? ELISA can not detect intracellular protien... I saw some signaling pathway proteins detected by ELISA

On a practical note, both techniques are used for protein detection.

However, i believe, the means by which they detect the target protein is different; western blotting separates proteins based on structure, before identification via antibody staining.

The last step is missing in ELISA test.

Which technique would be used would depend on the sample, protein of interest, and required accuracy of the results.

Hi, sorry for rehashing an old thread but does anyone have any references for these claims? I'm in agreement of that the use of the two are quite split with ELISA being better suited for quantification and WB for quality control, but I cannot find an article that supports me.

ELISA assays are based upon the principle of antibody/antigen binding. They enable quantification and characterization of specific analytes and/or molecular interactions.

Antibodies against the target of interest are conjugated to a reporter enzyme. Upon addition of its substrate, the enzyme catalyzes the production of a colorimetric molecule. The extent of this reaction is measured using a spectrophotometer and is representative of antigen concentration within a sample.

Western blots are often used to determine relative protein levels between samples. They also establish the molecular weight of the target, which may provide insight into its post-translational processing.

Proteins from tissue/cell lysates are separated by gel electrophoresis according to their molecular weight. Following separation samples are transferred to a membrane, where antibodies are applied to probe the protein of interest.