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Ab-Capcher Series/Kit Frequently Asked Questions


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Q:    I wonder if there is support protocol available for immunoprecipitation.
 
A:   Unfortunately, we do not have any protocol for IP. However, the protocol for GE protein A or protein G can be used. Please switch protein A (or protein G) to Ab-Capcher.
 
     
Q:   What percentage of gel slurry do manufacturer offer? I found Ab-Capcher-ExTra lately released is offered with 50% gel slurry in the manual. However, I couldn’t find that kind of information for Ab-Capcher to expect volumes to be taken.
 
A:   Ab-Capcher is also 50% gel slurry.
     
Q:   It appears that Ab-Rapid PuRe and Ab-Rapid SPiN are more suitable for micro-scale experiments. However, we have a customer who is interested in purifying secreted IgG from mammalian cells or bacteria culture medium (more than 1 L). I wonder if you have such references or recommendation guides. In this case, would you please check what size of column they need to prepare and how many volumes of gel bed should be packed in the column at the beginning?  
A:   We cannot say the detail things without any information of estimated concentration of IgG included in culture medium. 
    However, for your reference, we show one example below…
    --------------------------------------
    Mouse IgG1
    Concentration 0.1 mg/mL culture
    total volume 0.1 x 1000 = 100 mg
     
    Ab-Capcher ExTra
    Binding capacity: mouse IgG1, approx. 15 mg/mL
     
    Fill the column with 10 mL of Ab-Capcher ExTra, and pour Mouse IgG into it with flow rate 2mL/min. 
    Approx. 100 mg of IgG1 can bind to Ab-Capcher ExTra.
     
    In case human IgG, 2 mL of Ab-Capcher ExTra is enough to purify.
    Please note that binding capacity will be low with quick pouring of IgG.
    --------------------------------------
     
Q:   Do you know what parameters the customer can tweak to optimize the concentration? I’m sure he can probably do his own experimentation, but I don’t know if you or the manufacturer encountered his questions before or want to suggest anything? For example, can he do the things that he suggested below?   
    "I am trying to purify/concentrate mouse mab (most of them are IgG1) from commercial sources.  Usually, concentrations of those mans are around 200ng/ml and comes in 500ul -1ml and contains stabilizers such as gelatin, BSA and so forth.  I'd like to concentrate them to somewhere between 0.5mg/ml and 1mg/ml, ideal for downstream  fluorescent conjugation.  Ab-Rapid spin's max column volume is 0.8ml . If a total volume of mab solution is greater than 0.8ml  I have to use the same column and spin twice.  Is this possible ?  Also, recommended elusion volume is 200ul.  Is it possible to go lower like 100ul and is it necessary to spin more than once to get maximum amount of eluted mab from the column ? "
    1) Run a larger volume sample through the column twice?
    2) Reduce the elusion volume?
A:   For him, we recommend Ab-Rapid SPiN EX (#PTN-P-014-10). Powerful one!!
    1) Run a larger volume sample through the column twice?
    Yes. This product can accept duplicate apply of the sample.
    2) Reduce the elusion volume?
    Unfortunately, he CAN'T. The volume of elution buffer should be 200 ul.
     
    If the customer really needs to use 100 ul of elution buffer, there is another way.
    Mix Ab-Rapid SPiN EX (#PTN-P-014-10) well before use, and then, remove 200 ul of gel from the column to be 50 ul of gel in the column*. This column containing 50 ul of gel enables to use half volume of solutions for all the step (of course, including elution buffer). 50 ul gel can bind with 1 to 2 mg of mouse IgG1, so it's enough to his experiment.
    * The gel is 25% slurry. So, if 200 ul is removed, it will be 200 ul including 50 ul of gel.
     
    As mentioned is the data sheet,  if higher concentration of IgG is needed, 1st eluate can be use (it means twice eluate is not necessarily).
     
    Please note the following if the concentration of customer's sample is low;
    The concentration of IgG should be more that 0.1 mg/ml after purification because stabilizing reagent is removed by the usage of this product. If the concentration is too low, recovery rate will be low.
     
Q:   Can you please tell me if the AbCapcher resin is suitable for capturing IgM? 
A:   Some IgM can bind to Ab-Capcher, but some IgM cannot bind to Ab-Capcher. 
    So, we cannot say anything without checking. Please check your IgM whether it can bind to Ab-Capcher or not.
    Binding ability of IgM is weak compared to that of IgG. So, please elongate the incubation time (binding time) at Step 2 in our manual (e.g. 1 to 2 hours).

     
Q:   1) For how much refinement of the serum can Ab-Rapid Pure 2 (#P-012-2) be used? Also, please tell me the capacity of the column of this product and the capacity of filled Ab-Capcher.
    2) For how much refinement of the serum can Ab-Capcheri#P-002-2, 2MLj be used? 
    3) Please tell me the capacity of Buffer kit for Ab-Rapid Pure (#P-011). iThe registration size is 10RXNj
    4) Ab-Capcher is combined with rabbit IgG well. Is there any application data of the rabbit?
iAs there is the example of the mouse on a data sheet, I want to see it if there is a similar thing in rabbits.j

A:   1) Unfortunately we do not have the data with the rabbit serum. We have the voice of users that they obtained a satisfactory result by the IgG refinement from rabbit serum, using Ab-Capcher.




    Ab-Rapid Pure is filled with 0.5mL of Ab-Capcher.

    About 30 mg of human IgG is being combined and refined with syringe solution sending(0.3 mL/min) by using one column.
    As there was a case where 17 mg of IgG has been refined with Ab-Capcher 1mL from rabbit serum 5mL, about 8 mg could be combined by Rapid PuRe 0.5mL. But when you drain a liquid with a syringe, the purified quantity should be the half. Therefore, approximately 4 mg of rabbit IgG can be refined by one Rapid PuRe.
    Since in one example approximately 10 mg/mL of IgG is included in rabbit serum, approximately 4 mg (80% of yield) of IgG can be purified when 0.5 mL of serum has been diluted 3-5 times and then added to the Rapid PuRe, washed and eluted.
    Numerically, 0.4mL of serum is maximum.
    2) Ab-Capcher is being used for Rapid PuRe as well. Thus, the answer is the same as the answer 1).
    3) Combination bufferiPBSj@200 mL

    Elution bufferiGlycine-HCl, pH 2.8)  30 mL 
    Neutralization bufferi1 M Tris)    1 mL 
    4)  The answer is the same as the answer 1) and 2). Unfortunately, we do not have the application data of rabbit. We had an experience to purify 17 mg of IgG from rabbit serum using 1 mL of Ab-Capcher.
     
Q:   Can I get a free sample?
A:   We do not offer the free samples.
     
Q:   I would like to know about Ab-Capcher. I hear that antibody can be purified with high efficiency in comparison with conventional proteinA and G. I want to examine availability, so please tell me articles that introduce proteinA-R28 used.
A:   Since Ab-Capcher is affinity gel which has been  released recently, there are no articles yet that the results using it are listed in.

 
   

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


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