advice on activation of PEG-Carboxylic Acid with EDC and sulfo-NHS?

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marine2008
marine2008's picture
advice on activation of PEG-Carboxylic Acid with EDC and sulfo-NHS?

My target is to conjugate about 150 kDa IgG Antibody to PEG micelles via the activation of PEG carboxylic acid with EDC and sulfo-NHS. Ab: DSPE-PEG(2000)Carboxylic ammonium (Avanti) (1:40, molar ratio) was adapted in my test. But I have no idea on the assay of the reaction products and am also wonder if 1) the ratio is in an appropriate range for effective conjugation, 2) I can add weighed EDC and sulfo-NHS powder directly to the DSPE-PEG2000 carboxylic ammonium in MES, pH 5. Can anybody give advice? Thanks a lot.

Hatuey
Hatuey's picture
Hi, My suggestion is to mix

Hi, My suggestion is to mix first the carboxylic acid PEG withthe EDC and NHS-sulphoacetate and then add the protein to the mix after few seconds, ensure you mix well everything. the ratio of the components should be something like this : carboxylic acid-PEG/EDC-sulphoNHS/protein:  10:100:1, in such a way that you have an excess of the PEG over protein, now the risk of this is that you could get some crosslinking of the protein with itself so in this case you should reduce the concentration or qty of the EDC-sulphoNHS, this is quite empirical some proteins migh not crosslink withthenmselves too much depending onthe qty and availability of the -NH2 and -COOHs inthe protein, so you should try different ratios and run gels to determine which ratio is better. The reaction should be incubated for short time, maybe not more than one h to decrease the crosslinking . All this is very empirical and depends onthe nature of the protein, also if you can you could separate the activated PEG fromthe excess of EDC-NHSacetate before adding the protein, for example, using a microcon filter, this is the best, but not always is possible, depending on the MW of the PEG;  these are the general guidelines for this kind of conjugation.

marine2008
marine2008's picture
Thank you so much for kind

Thank you so much for kind suggestion. Actually I added the protein 15 minutes after mixing of carboxylic PEG, EDC and sulfo-NHS. But the conjugation efficiency was not as high as that of reported similar work. I am going to do the experiment again following you suggestion and hope to see great result. Best regards.

marine2008
marine2008's picture
I have another question about

I have another question about the use of EDC and sulfo-NHS. Can they still be useful to activate carboxlic acid if the buffer contains ammonium ion? I wonder whether ammonium is an interference. I'm eager to get help! 

sam60
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Dear marine

Dear marine
Did you find the best solution for the conjugation of antibody to PEGylated liposome?
Let me know about this.

blair
blair's picture
Dear Marine2008:

Dear Marine2008:
 
Glad to write to you!
 
This is Ningbo Roger Biotech Co., Limited (RG BIOTECH).We dedicate to design, develop and distribute PEG linkers(mPEG-SC,mPEG-SPA,mPEG-MAL,mPEG-ALD,ETC.) with GOOD QUALITY and COMPETITIVE PRICE.
 
Kindly contact me if any questions. It is our pleasure to be on service of you!
 
Thanks and best regards,
 
Miss Blair
 
Ningbo Roger Biotech Co., Limited (RG BIOTECH).
Add:No.128,YingchunRoad,Gaoqiao,Yinzhou District,Ningbo,China
Tel:+86-574-87740578
Fax:+86-574-87740558
Mail: eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%62%6c%61%69%72%67%6d%6d%40%79%61%68%6f%6f%2e%63%6e%22%3e%62%6c%61%69%72%67%6d%6d%40%79%61%68%6f%6f%2e%63%6e%3c%2f%61%3e%27%29%3b'))
Web:http://www.creatchemicals.com/

blair
blair's picture
Dear Hatuey:

Dear Hatuey:
 
Glad to write to you!
 
This is Ningbo Roger Biotech Co., Limited (RG BIOTECH).We dedicate to design, develop and distribute PEG linkers(mPEG-SC,mPEG-SPA,mPEG-MAL,mPEG-ALD,ETC.) with GOOD QUALITY and COMPETITIVE PRICE.
 
Kindly contact me if any questions. It is our pleasure to be on service of you!
 
Thanks and best regards,
 
Miss Blair
 
Ningbo Roger Biotech Co., Limited (RG BIOTECH).
Add:No.128,YingchunRoad,Gaoqiao,Yinzhou District,Ningbo,China
Tel:+86-574-87740578
Fax:+86-574-87740558
Mail: eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%62%6c%61%69%72%67%6d%6d%40%79%61%68%6f%6f%2e%63%6e%22%3e%62%6c%61%69%72%67%6d%6d%40%79%61%68%6f%6f%2e%63%6e%3c%2f%61%3e%27%29%3b'))
Web:http://www.creatchemicals.com/