14 replies to this topic

[Adrian K]

Hi All,
I had cloned 5 gene of interest in pet46EK/LIC and expressed in E. coli BL21(DE3) (Novagen)
The vector will produce 6x his-tag in the front part of the protein.

I had expressed the protein, run SDS-PAGE, and perform western blot.
I use the KPL HisDetector Western Blot Kit, AP Colorimetric, Cat No 25-00-01 https://www.kpl.com/...heet/250001.pdf
for my his-tagged detection.

However, i do encounter few issues:

1. Consistent background protein detected on ~25kDa
------ Do you experience such problem with BL21(DE3)?
------ The background appeared higher than my protein of interest.

2. Inconsistent background protein detected at ~32kDa, only in UREA treated lysate, B.


3. Possible overlapped Recombinant 2 with background on lane B, Recombinant 2?


The PDF I attached had complete descriptions about my problem.
Perhaps you might have an idea on where goes wrong. I really need help in troubleshooting as I got no idea what went wrong...

Million thanks in advance.

Adrian

Attached Thumbnails

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Attached Files

•  for forum WB 21032011.pdf   77.6K   113 downloads

Edited by adrian kohsf, 21 March 2011 - 08:11 PM.

[mdfenko]

what you are referring to as "background" would be called "non-specific binding" if you were using an antibody for detection. since you are detecting by binding nickel you are seeing nickel binding proteins, presumably his-tagged proteins but not necessarily.

some of them may be co-migrating with (overlapping) your proteins of interest.

you can try separating them by ion exchange in the presence of urea.

[pDNA]

the first thing i see from your gels is that the SMART Protein Extraction Solution that you used does not perform very well
not a good advertisement ...the good old classic approach does outperform this for sure!

To me it seems like mdfenke mentioned unspecific binding ...the band at above 24 kDa is strange since it is everywhere so it must be some E. coli protein that binds to that antibody or another artefact from somewhere ...maybe you can think about how you prepared the samples for the PAGE because it is strange that this protein seems to be in all fractions ...maybe your loading buffer is contaminated?

The bands in the lysate ( might be due to some agens in the lysis buffer? ...do you used lysozyme? Lysozyme is 14 kDa in size ...that would fit to the lower band. The higher the protein concentration of a band the more it tends to bind unspecific to the antibody ...we often see that with lysozyme ...if there is no protein at all you will get for sure a signal for the lysozyme that we use for cell lysis if you only incubate long enough with BCIP for example.

I don't know how you stained that western but maybe the additional bands are just artefacts and everything is fine!

Regards,
p

[Adrian K]

mdfenko, on 22 March 2011 - 08:10 AM, said:

what you are referring to as "background" would be called "non-specific binding" if you were using an antibody for detection. since you are detecting by binding nickel you are seeing nickel binding proteins, presumably his-tagged proteins but not necessarily.

some of them may be co-migrating with (overlapping) your proteins of interest.

you can try separating them by ion exchange in the presence of urea.

Hi mdfenko,
Thanks for your reply. I do not have ion exchange system, is there any other way that I can possible go around?

pDNA, on 22 March 2011 - 09:49 AM, said:

the first thing i see from your gels is that the SMART Protein Extraction Solution that you used does not perform very well
not a good advertisement ...the good old classic approach does outperform this for sure!

To me it seems like mdfenke mentioned unspecific binding ...the band at above 24 kDa is strange since it is everywhere so it must be some E. coli protein that binds to that antibody or another artefact from somewhere ...maybe you can think about how you prepared the samples for the PAGE because it is strange that this protein seems to be in all fractions ...maybe your loading buffer is contaminated?

The bands in the lysate ( might be due to some agens in the lysis buffer? ...do you used lysozyme? Lysozyme is 14 kDa in size ...that would fit to the lower band. The higher the protein concentration of a band the more it tends to bind unspecific to the antibody ...we often see that with lysozyme ...if there is no protein at all you will get for sure a signal for the lysozyme that we use for cell lysis if you only incubate long enough with BCIP for example.

I don't know how you stained that western but maybe the additional bands are just artefacts and everything is fine!

Regards,
p

Hi pDNA,
Thanks for your reply. Haha, I do not work for that SMART protein company, or maybe is my poor technique?  
Perhaps the consistent ~25kDa is some propriety agent used in the reagent itself. Maybe some strange chemical reaction happened when I boiled it with my loading buffer which cause it happen.

To rule out the SMART protein reagent and possible contamination from loading dye, I had run another gel this time, with one lane only consist of SDS-Loading Dye + water only. The rest is just SDS-Loading Dye + sample. I will restain the WB once i go back to my lab 6 hours later... is 3.30 am now... and lets see what happens later.

I was thinking the possibility of cross contamination from pipettor, or somebody had spiked my work...

Is there any good method for protein isolation, possible without using "benzamidine⋅HCl " or "guanidine⋅HCl"?

Many thanks to mdfenko and pDNA again.
Adrian

[mdfenko]

hydrophobic interaction?

[Adrian K]

Here is my new WB and silver stain. Totally without the SMART protein solution.
Yeah mdfenko, you are right. Those are probably nickel binding protein. I had excluded the possibility of my SDS buffer/loading buffer/water is contaminated as the negative lane shows clear. Thanks for pointing that out pDNA. I almost throw away my buffer which i newly bought.

In my attachment, I assume that in order for the western to detect the band, it must have certain concentration and visibility in silver stain.
But for the constant non-specific binding was not "seen" in the silver stain, this really troubles me.

Is there anyway which I might be able to get around it,by eliminate the non-specific binding of nickel?
Any idea?

@mdfenko, how to perform the hydrophobic interaction, any links for me?

Manby thanks.

Attached Files

•  Western Blot 23.pdf   118.28K   99 downloads

[mdfenko]

adrian kohsf, on 23 March 2011 - 07:26 PM, said:

Here is my new WB and silver stain. Totally without the SMART protein solution.
Yeah mdfenko, you are right. Those are probably nickel binding protein. I had excluded the possibility of my SDS buffer/loading buffer/water is contaminated as the negative lane shows clear. Thanks for pointing that out pDNA. I almost throw away my buffer which i newly bought.

In my attachment, I assume that in order for the western to detect the band, it must have certain concentration and visibility in silver stain.
But for the constant non-specific binding was not "seen" in the silver stain, this really troubles me.

some proteins don't stain well with silver, some require double staining. a neighbor of mine had to silver stain twice to see her protein.

Quote

Is there anyway which I might be able to get around it, by eliminate the non-specific binding of nickel?
Any idea?

you could use an antibody to the protein (or his-tag) instead of binding nickel.

Quote

@mdfenko, how to perform the hydrophobic interaction, any links for me?

how about this:

Attached Files

•  hydrophobic interaction.pdf   2.55MB   92 downloads

[Delfis]

Hi. I'm having the exact same problem : a 25KDa band from hell .Only I'm using Promega MagneHis kit to purify my his-tagged proteins. Please tell me if you found a solution to this problem! I've already ruled out the buffers/magnetic beads/etc and I'm out of ideas.

[Adrian K]

Hi Delfis,
I had eliminate the problem in WB lately, I managed to remove the 25KDa band in WB, although in silver stain, I still got some bands remain.
What I did was increase the in-column washing, combined with using different washing solution (one with b-mercapto, one with tween-20).
However I not sure whether your beads can survive in  buffer with b-mercapto.
I remember MagneHis kit did mentioned about adding NaCl for more specificity, but I have yet to try it yet.

Adrian

[Delfis]

Thank you so much for your answer. I just checked and MagneHis beads tolerate up to 100mM b-mercapto and 0.05% tween-20. How much did you use? I already use NaCl fo specifity, but I could try raising it even more. Thank you again!

[Adrian K]

Can't remember off hand the exact procedure because my notes still in the lab...
however briefly,
http://www.mn-net.co...otinoNi_TED.pdf

I am using Ptorino Ni-TED 1000 column, using the method for native protein, but I sonicate without lysozyme.
Proceed with manufacturer's protocol.

Washing: wash buffer (W- with b-mercaptoethanol 30mM, then W-B with tween-20 0.2% (Thanks to mdfenko for the tip ), then W-B with 1mM Imidazole, then followed by just normal W-B. Finally is elution.

In the elution, you got 3 elutions. I use the second elutions for majority of my proteins. If WB detects nothing in my second elutions, I go back to the first elution which appears to be more dirty (first elution is still with some 25kb band from hell, no choice)

Since my second elution is not concentrate enough (I guess...) I concentrate ot and buffer exchange to 0.1M HEPES buffer it with vivaspin (took ages waiting for the spinning). I do a-repurification later with MagneHis beads, without modification. IT took nearly 24 hours continuous working in the lab...

The result: I got a single clean band in WB, but in silver stain, I still get 4 bands in background. Not sure what caused it.

If you are from Malaysia we can talk over the phone and do some further discussions, perhaps meet up and maybe a mini-collaboration to make the protein purification in final: 1 band in WB, 1 band in silver stain...

Regards,
Adrian

Edited by Adrian K, 06 September 2011 - 06:03 PM.

[Delfis]

Well, thank you very much for all the details! i'm going to try to incorporate these changes to my system. I hope this finally solves the problem! Thanks again!

[Adrian K]

Do let us know the outcome

[Delfis]

Hi, I`m back. I´ve tried doing washes with both b-mercaptoethanol and  tween-20 (in separate washes)...and there was still the band from hell in coomasie stain (I didn't try WB, since I already saw it in the gel). I also saw the same band in the non-IPTG induced control. Granted, the band is quite faint, specially in comparison to the band of my protein, but I'm really trying for more purity (maybe that is the best I'll get...though I can't understand why I din't see it before). It does seem that there is some  histidine rich-protein from e.coli I just can't get rid off. So...I'm sorry to be bothering again, but does anyone have any other ideas?. Anything will help. Thanks!

[Adrian K]

For me, I still got the phantom bands in my silver stain (although my protocols were different with yours) but it doesn't come out in WB.. so I think is fine for me...

Maybe you can try column purification such as FPLC, size exclusion etc...