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problem with metal conjugation

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komalkumaralienboy

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Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Wed Jul 15, 2015 6:43 am

problem with metal conjugation

Helllo ,

I did metal conjugation yesterday for 3 intercellular proteins. After conjugation , Nanodrop showed 85mg/ml for 1 protein but for the remaining 2 antibodies showed negative results.

Purified antibodies from Cell signaling Technology. I used Maxpar metal conjugation protocol. finally antibodies stored in protein stabilizer buffer.
TCEP prepared freshly.

Any suggestions
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robem01

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Post Wed Jul 15, 2015 6:02 pm

Re: problem with metal conjugation

Hi Komal, We had a similar issue here. Some antibodies had a decent recovery where others did not. We've switched to 30K filters in the conjugation instead of 50K filters and this switch in filter size has allowed us to recover all antibodies! Hope that helps.

-Emily
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HRsumatoh

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Location: Singapore

Post Thu Jul 16, 2015 3:30 am

Re: problem with metal conjugation

Hi Komal

I hope your purified antibodies do not contain any glycerol, gelatin or any other stabilizers that might affect the conjugation process.

Cheers

Rizal
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Thu Jul 16, 2015 6:20 am

Re: problem with metal conjugation

Hi,
ooppppps

Hi Rizal,

Yes storage buffer contains BSA , Glecerol, sodium HEPES, Nacl.

do u have protocol to remove the above ingredients from the purified antibodies.
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mleipold

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Location: Stanford HIMC, CA, USA

Post Thu Jul 16, 2015 3:00 pm

Re: problem with metal conjugation

Glycerol, trehalose, and buffer salts do not interfere with the conjugation reaction (antibody disulfides being cleaved by TCEP to generate free Cys thiols, which then react with the maleimide at the terminus of the metal-loaded polymer).

Gelatin does not contain any Cys, therefore will not interfere with the conjugation reaction either. However, it will usually prevent you from getting an accurate Nanodop A280 reading for your protein yield, as it is also a protein.


Any thiol-based reducing agent (DTT, b-mercaptoethanol, etc) will severely reduce the conjugation efficiency. However, it should not affect the Nanodrop reading.


BSA will severely interfere with the conjugation reaction. BSA has 33 Cys: typically, 1 Cys is free, while 32 are involved in forming 16 disulfide bridges. Once that sees TCEP, some or most of them will break, and will generally outcompete your reduced antibody for reaction with the polymer maleimide. Additionally, like gelatin, BSA will interfere with proper Nanodrop protein recovery determination.


There are BSA removal kits on the market (search on Cytoforum for some recommendations, as this has been a topic in the past). However, the best option, if possible, is just not to use BSA-containing antibody stocks.....it's often worth the extra expense for a custom prep without BSA.


Mike
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billog

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Joined: Wed Sep 17, 2014 10:53 pm

Post Thu Jul 16, 2015 5:04 pm

Re: problem with metal conjugation

With Gelatin containing mAbs you might try conjugating less than 100ug's if your 100ug conjugation turns to jello at 4oC.... I've had this happen to me when the gelatin concentration was initially sufficiently high to cause aggregation in my final conjugate.

This could be a useful list:

BD: usually purified is available (for now!), but otherwise BSA
Biolegend: usually purified is available, but otherwise BSA
eBio: sometimes purified is available, but otherwise BSA (I think)
CST: glycerol; need to special order purified
R&D: usually Trehalose- not a problem
Santa Cruz: usually Gelatin; easy to get special order purified (relative to other companies)
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jul 16, 2015 5:30 pm

Re: problem with metal conjugation

Hi Bill,

Regarding your CST entry: does CST usually have something in addition to glycerol?

I'm puzzled why you're special-ordering if there's only glycerol as an additive (rather than BSA/gelatin/other carrier protein).


-Mike
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billog

Participant

Posts: 15

Joined: Wed Sep 17, 2014 10:53 pm

Post Thu Jul 16, 2015 8:42 pm

Re: problem with metal conjugation

CST: Glycerol + 100ug/ml BSA

Sorry I omitted the BSA for CST

Also good to say any mAb company whose QC consists of end user reviews is best avoided altogether regardless of cytometry platform...

CST is very rigorous in its mAB QC...
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Fri Jul 17, 2015 5:54 am

Re: problem with metal conjugation

HI,

I ran the samples before conjugation and after conjugation in flowcytometry to confirm the protein signal. the signal intensity was reduced very much after conjugation.

for example before conjugation MFI was 6000, after conjugation it was 2000.
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AdeebR

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Location: NYC

Post Fri Jul 17, 2015 1:56 pm

Re: problem with metal conjugation

robem01 wrote:Hi Komal, We had a similar issue here. Some antibodies had a decent recovery where others did not. We've switched to 30K filters in the conjugation instead of 50K filters and this switch in filter size has allowed us to recover all antibodies! Hope that helps.

-Emily


Hi Emily,
That's a really helpful tip. I've had the same experience with variable antibody recoveries - just last week I conjugated 14 antibodies, all of which gave me recoveries in the 75-85% range except for one that was ~25% (anti-CD64 clone 10.1).

Just to clarify, did you conjugate an antibody with a 50K filter and get a low recovery and then re-conjugate the same antibody clone with a 30K filter and get a higher recovery?

Would there be any concerns that would warrant not to use a 30K filter for all conjugations?

komalkumaralienboy wrote:HI,

I ran the samples before conjugation and after conjugation in flowcytometry to confirm the protein signal. the signal intensity was reduced very much after conjugation.

for example before conjugation MFI was 6000, after conjugation it was 2000.


Hi Komal,

Could you clarify how you were testing your antibody by flow cytometry? Were you detecting it with an anti-isotype secondary? Or were you metal-conjugating a fluorescently-labeled primary?

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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