Hi guys,
I am using the papain digestion system to abtain a Fab.
Here is my recent result, I got thin bands around 55, 45; thick bands around 30, 25, which may indicate incomplete digestion, but what bothers me is the 11 kDa band here, appeared in both reducing (left) and non-reducing (right) condition.
Here is my digestion system:
50 mM Sodium phosphate buffer
10mM Cystein
Sample application buffer 4x, sample/SAB (v/v)=3:1
4h digestion with ratio IgG1: Papain=50:1
Any suggestions would be greatly appreciated.
Hi,
Before papain cleavage, Are you also observing this 11 kDa band at intact IgG1 SDS-PAGE analysis?
Is the purity of your antibody sufficient for SDS-PAGE but also for papain activity?
Perhaps the pieces of information below would be helpful for buffer compositions and procedures (ratio & concentrations, incubation conditions etc.) at your application...
Mercuripapain is pre-activated with 10mM cysteine in 1.25mM EDTA for 15 minutes at 37C, and then added to the IgG (5-10 mg/ml) at a 1:50 to 1:200 (w/w) ratio enzyme to antibody. A series of incubations are carried out at 37C for 15min-12 hours to determine the susceptibility of the antibody to cleavage. The cleavage is stopped by the addition of iodoacetamide (20-50 mM). Optimal conditions are determined by analysis of the products of the enzymatic cleavage by SDS-PAGE.
Good Luck,
Emir.
Hi,
Before papain cleavage, Are you also observing this 11 kDa band at intact IgG1 SDS-PAGE analysis?
Is the purity of your antibody sufficient for SDS-PAGE but also for papain activity?
Perhaps the pieces of information below would be helpful for buffer compositions and procedures (ratio & concentrations, incubation conditions etc.) at your application...
Mercuripapain is pre-activated with 10mM cysteine in 1.25mM EDTA for 15 minutes at 37C, and then added to the IgG (5-10 mg/ml) at a 1:50 to 1:200 (w/w) ratio enzyme to antibody. A series of incubations are carried out at 37C for 15min-12 hours to determine the susceptibility of the antibody to cleavage. The cleavage is stopped by the addition of iodoacetamide (20-50 mM). Optimal conditions are determined by analysis of the products of the enzymatic cleavage by SDS-PAGE.
Good Luck,
Emir.
Are you sure the 11-kDa band is not another digestion product, since appear also in non-reducing conditions? Has you tried to repeat the assay with another IgG and papain preparations?
Ruojin Tian As İsmail Emir Akyildiz and María Luisa Caballero said above, check your sample in advance. And you haven't reduced SDS-PAGE samples after digestion, right? Even if the samples were not heated, the reduction might be promoted slightly by the DTT contained in the sample buffer. Do you use sample buffer without DTT or other reducing reagent? I hope this guess is wrong. It is better to show all the gel-image as people cannot determine whether "thin bands you think" is appropriate.
Michitoshi Watanabe First of all, thank you for your generous answer!
I have done with ddt+heating and no ddt+no heating.
One more update: After I diluted the sample with MES buffer (for purification), the band around 11 kDa was gone. It seems like it recovered in the buffer.
I think I will check the digestion by other IgG or send to the company to check about the sequence of it.
Have a nice day!
María Luisa Caballero Thank you for your kind suggestion!
I think I will check the digestion by other IgG or send to the company to check about the sequence of it.
BTW, one more update: After I diluted the sample with MES buffer (for purification), the band around 11 kDa was gone. It seems like it recovered in the buffer.
Have a nice day!
İsmail Emir Akyildiz Hi, thank you for your kind respond!
In intact IgG, there is no such a band around 11 kDa.
The purity and the papain activity are also good.
I think I will check the digestion by other IgG or send to the company to check about the sequence of it.
BTW, one more update: After I diluted the sample with MES buffer (for purification), the band around 11 kDa was gone. It seems like it recovered in the buffer.
Have a nice day!
I agree Dr Watanabe on the suggestion to show the entire gel to better understanding the problem.
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