Hello!
I am struggling with Western blot. I would like to measure protein levels in bacteria cells, so I used western blot technique. Onto gel, I loaded bacteria lysates and the purified protein of interest (his-tagged). I performed WB. The purified protein is recognized by antibodies and has proper molecular weight compared to molecular mass marker, however, native proteins in cell lysates migrate much slower. Bacteria are lysed in Laemlie buffer and boiled at 95C before SDS-PAGE electrophoresis. I do not separate soluble and insoluble fractions of cells or digest DNA.
What could be a possible explanation for this? is it possible that somehow crude extract slowed down protein migration in SDS-PAGE?
I believe that the antibodies, which I used work properly - they recognize purified proteins and work well in ChIP. In the attachments, I loaded the WB result.
This looks like proteolysis. Another possibility is that the protein forms a very stable complex that survives heating in Laemli sample buffer (rare, but not unheard of). Difficult to tell since it is unclear how big the mass difference betwen the two bands is.
Dear Ewelina Wysocka
There can be different possibilities regarding the difference in the protein migration.
- There might be intermolecular disulphide bonds formed. If your Laemmli buffer contains reducing agent (2-mercaptoethanol or DTT), you can omit this problem.
- The protein of interest can be a DNA binding protein. If so, then the cell lysis should be performed in presence of Benzonase or DNase.
- If the protein of interest is soluble, collect the supernatant followed by lysis.
Best wishes.
Engelbert Buxbaum thank you for your answer. I am hurrying up with the explanation. The protein of interest has 52 kDa (regarding AA sequence), purified protein migrates near the 55 kDa marker band (lane 7), however, the band in bacteria lysate is nearer 70 kDa marker band (lanes 1-5), so the mass of the protein recognized by antibodies in lysate is about 15 kDa bigger than purified protein/ molar mass.
Satyendra Mondal I use 2-mercaptoethanol in Laemmli buffer. This protein is a DNA binding protein, but I would expect a higher change of mass if its a DNA-protein complex. Nevertheless, I will try with DNAse. Thank you!
Given it is a DNA-binding complex, you could try lysing in the presence of EDTA, or another chelating agent: this could possible liberate the DNA.
That said, it depends what you are trying to achieve here.
What is the source of your purified protein? Is it protected from proteolysis during its purification? Is it the same construct (a.a. composition)?
Another method could be to provide DNA to your purified protein and see if you can achieve the reverse effect: an electrophoretic mobility-shift assay.
Other options:
try heating at 70°C for 10 min;
DTT or TCEP as reducing agent;
run samples without boiling to see if the purified band has a different mobility.
Proteins in crude extracts typically contain a mixture of different proteins, including impurities and other non-target proteins, which can interfere with protein migration during SDS-PAGE. Additionally, crude extracts can contain proteins that are not fully denatured, which can also affect their migration. These factors can cause crude extract proteins to migrate slower than purified proteins during SDS-PAGE.
On the other hand, purified proteins are typically of higher purity and are fully denatured, which allows for more uniform protein migration during SDS-PAGE. The removal of impurities and non-target proteins can help eliminate any interference with protein migration. Additionally, purified proteins can be more concentrated, which can also help ensure consistent protein migration.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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