[Johan Rojek]

Hello everybody,

I’m having trouble enriching biotinylated proteins using Dynabeads MyOne streptavidin C1. If somebody has experience working with streptavidin beads or is able to spot any wrongdoing on my part, I will appreciate any advice.

I have been testing whether the beads can be used to capture and elute biotinylated proteins obtained from a CHO cell culture expressing a biotinylase. The problem is that the beads seem to capture non-biotinylated proteins.

Before the Dynabead enrichment, I examined my samples with a streptavidin-HRP western blot. The blot showed a clear distinction between the biotinylated samples and the wildtype control.

Western blot and gel picture:

View post on imgur.com

However, when I subsequently tested the Dynabead enrichment, proteins were eluted from the wildtype sample as well. Furthermore, the protein band profile of the wildtype eluate was similar to the samples containing biotinylated proteins.
Another curious observation is that the protein band profile is not comparable to the positive samples in the western blot.

Specifics:

For enrichment of biotinylated proteins:
The beads were washed twice in DPBS and incubated with the lysate samples for 1 hour with rotation at room temperature and subsequently at 4 degrees with rotation O/N.

The wash steps:
2x DPBS
1x 1M KCl
1x 0.1M Na2CO3
1x 2M Urea in 50 mM Tris-HCl (pH 7.5)
2x DPBS

Elution:
Boiling the beads in NuPAGE™ LDS Sample Buffer and NuPAGE™ Sample Reducing Agent supplemented with 2 mM biotin (for competitive binding).

Additional notes:
– The elution has been repeated a total of three times with similar results.
– From the second PBS wash step and onward no protein observed when being run on a gel (I, unfortunately, did not sample the first PBS wash..)
– The Western blot was been repeated to test if the different protein band profiles were caused by degradation over time. However, the blot was identical to the first.
– In case you are wondering why the biotinylase works without supplementation of biotin, the commercial media used already contains an unknown concentration of biotin.

Best regards,
Johan

• This topic was modified 8 months, 3 weeks ago by Johan Rojek.
• This topic was modified 8 months, 3 weeks ago by Johan Rojek.

[Nikolay Dobrev]

Dear Johan,
Can I ask which BirA variant you are using?
I assume that your protein has an Avi tag, am I right?

I did not see also what is exactly the size of your protein?

May be I did not get something, but why you are concluding the following:

“The problem is that the beads seem to capture non-biotinylated proteins”

Best
Nikolay

[Johan Rojek]

Dear Nikolay,

Thanks for the reply.

I’m testing the TurboID BirA variant.
Biotinylase 1 is a given bait-protein fused with the TurboID biotinylase (about 90 kDa). While Biotinylase 2 is just TurboID (35 kDa).

Neither Biotinylase 1 nor 2 have Avi tags.
I’m interested in identifying endogenous proteins that are labeled with biotin from being in close proximity to the TurboID enzyme.

It seems to me that the Dynabeads bind a range of non-biotinylated proteins based on the WT sample (negative control) eluate run on the Coomassie-stained gel.
Based on the Western blot, the WT sample should only two bands (of about 80 and 150 kDa).

-Johan

[Nikolay Dobrev]

Dear Johan,
Now, I understand! You are performing BioID.

I will come back to you with some suggestions.

Best,
Nikolay

[Krzysztof Skowronek]

Dear Johan,
According to my experience there is quite a lot of proteins that can bind unspecifically to any kind of paramagnetic (or even agarose) beads. I guess the best way is to preclear your lysates first on similar beads but without streptavidin. For instance you can use amine Dynabeads (https://www.thermofisher.com/order/catalog/product/14307D#/14307D) as I guess this is what Thermo is using to produce Streptavidin Dynabeads. So you should expose your extracts first to amine Dynabeads and then supernatant can be transfer to Streptavidin Dynabead for “more specific” capture of biotinylated fraction.

Best,

Krzysztof

[Johan Rojek]

Dear Krzysztof,

Thank you for the input.
It is valuable to know that non-specific protein binding can occur when using beads. I was hoping my wash steps would be suitable to disrupt the weaker bindings. This may cause me to approach the situation differently.

I will look into the possibility of pre-clearing the lysate on similar beads without streptavidin.

Best,
Johan

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