2
$\begingroup$

So I was in class and my professor was explaining western blotting. It seems as though there's more than one way to increase or decrease the signal intensity. He presented us the challenge to discover different ways this can happen. As hard as I look all I can find on the internet or even in google scholar is just certain chemicals like 'signal enhancer' and that's it, nothing remotely specific. Legitimately I do not propose this as actual homework, in fact it's just a curiosity I have. Therefore I ask the scientific community what, if any, are conditions that one could increase or decrease the signal intensity in western blotting? I understand what western blotting is, anyone can find this on wikipedia, but conditions that could increase or decrease signal intensity?

$\endgroup$

3 Answers 3

2
$\begingroup$

There are several different places in a typical Western blotting protocol where conditions can be altered to modulate signal intensity. The first is the amount of protein loaded - more protein will give a stronger signal, less will give a weaker signal. However, too much protein can result in indistinct bands, increased background, lane distortion, and other issues.

The next condition to change is the amount of primary antibody and the incubation buffer. More primary may lead to an increased signal, but too much can give higher background. Some antibodies give a better signal when diluted in BSA, others when diluted in nonfat dry milk. Generally, milk will give you a cleaner blot, but may reduce the signal. Incubation time can also affect specificity and signal intensity - some antibodies will fully bind with a 1-2 hour incubation at room temperature, others require an overnight incubation at 4°C.

The third condition to alter is the amount and type of secondary antibody. Increasing the amount of secondary may increase signal, but again may result in increased background. There are also different types of secondaries, depending on your detection system - HRP for chemiluminescent detection, AP for colorimetric, fluorescent tags for imaging systems, etc. These have different sensitivities, but can also result in increased background, depending on the other conditions of the protocol.

Finally, the type of detection system can affect sensitivity. For example, when using standard HRP-conjugated secondaries, there are numerous different ECL substrates with widely varying sensitivities. Here is a list of different systems available from Thermo/Pierce, ranging from standard ECL to Femto.

So, while Western blotting seems like a fairly straightforward procedure, there are many places to optimize the conditions to get the results you're looking for.

$\endgroup$
3
  • $\begingroup$ Wow I was sort of close with some of this yet so far away. That is amazing, you must work with western blot on a regular basis? Curiously, could I get a reference from you as to where or how you got this? $\endgroup$
    – Fistula
    Dec 7, 2014 at 21:30
  • $\begingroup$ @Fistula I worked for an antibody company for over 6 years doing antibody preparation, QC, and tech support, so this answer is just a quick distillation of my knowledge. I can talk for hours about Westerns, if you're so inclined :) $\endgroup$
    – MattDMo
    Dec 7, 2014 at 22:31
  • $\begingroup$ This is a good answer @Fistula $\endgroup$
    – rhill45
    Dec 12, 2014 at 1:02
2
$\begingroup$

One thing that others have not mentioned yet is that the time of the wash can affect the signal. If its washed longer than the usually required 5 minute three time wash, this could result in a reduced (decreased) signal.

Reference: http://www.cellsignal.com/common/content/content.jsp?id=western-trouble

$\endgroup$
1
$\begingroup$

Additionally to the points in the answer @mattdmo has given, it is possible to enrich the protein of choice before the gel run. This can either be done by using tagged proteins (possibly with the cleavage of the tag before loading) or by doing an immunoprecipitation of your protein. The later is interesting when you have only low concentrations of your protein present in the cell. What you basically do is to bind an antibody to the protein, the bind the protein-antibody-complex to a column or beads, wash away (in theory, practically you are not getting all the other proteins away) and then load the sample. Depending on the enrichment, you will be able to see a band, where this is not possible without immunoprecipitation.

Then washing and blocking of the blot plays an important role. Usually you use milk powder to prepare the blocking solution, as this is quite cheap and works pretty well. But it can give quite a high background and make problems with phosphoproteins (not necessarily though). Sometimes it's better to use more expensive solutions like BSA for blocking the membrane. The washing is also critical, depending on how stringent you wash, you will loose signal and influence the background. Here you can play with different concentrations of detergents as well as with different detergents.

Additionally you can get a lot of different signals in the detection. For chemolumincence based detection you can either use x-ray films (which are rather unsensitive) and have problems with strong bands outshining neighbouring weak ones. They are also harder to quantify, as the linear range in which more intensity corresponds with more signal is limited. With chemoluminescence you can also use special highly sensitive CCD-cameras (which are more sensitive and have a much higher linear detection range) or phospho-imager plates (special plates, which store the chemoluminecent signal on a light sensitive plate and which are read out with a special scanner).

The latest development in the detection technology are infrared scanners for which you use secondary antibodies coupled to a dye which fluoresces at infrared wavelength. These scanner are extremely sensitive and also allow the usage of two differently labelled secondary antibodies at the same time, which can be very handy in analyzing protein interactions. You will need primary antibodies from different species, though. The modern detection techniques also have the advantage of delivering digital files, while films need to be scanned before further use in publications.

$\endgroup$
1
  • $\begingroup$ Also a good answer @Fistula $\endgroup$
    – rhill45
    Dec 12, 2014 at 1:02

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .