4 replies to this topic

[Kansas313]

Hi All,

I need help in detecting a small protein (~15kD) with western blot. I believe that this protein has very low expression in plant tissues. I am able to detect the positive control (50 ng expressed protein) but not from the plant tissues. I load lot of protein on the 12% Bis-Tris gel (160 ug/well) from the plant tissues and transfer for 1.30 Hr at 21 V. Do blocking with 5% milk in TBST for 1 Hr and primary antibody incubation in 1 % milk at 4c overnight.

Please help me with any suggestions or protocol for my problem.

Thanks,

[than4]

Not sure about the low expression problem, but to detect a protein 15kDa in size I would be running a 15% gel.  I used to look for cytochrome c which was about the same size and always ran a 15%.  Your transfer time seems okay so as not to be transferring the small proteins through the membrane.  Could you try to concentrate your protein by doing an IP from the plant cells?

[fishdoc]

Kansas313, on May 17 2010, 10:51 AM, said:

Hi All,

I need help in detecting a small protein (~15kD) with western blot. I believe that this protein has very low expression in plant tissues. I am able to detect the positive control (50 ng expressed protein) but not from the plant tissues. I load lot of protein on the 12% Bis-Tris gel (160 ug/well) from the plant tissues and transfer for 1.30 Hr at 21 V. Do blocking with 5% milk in TBST for 1 Hr and primary antibody incubation in 1 % milk at 4c overnight.

Please help me with any suggestions or protocol for my problem.

Thanks,

You could try using a more sensitive chemiluminescent substrate (if you're using chemiluminescence). I'm assuming you're using a conjugated secondary. You could try using a biotinylated secondary, then use a HRP-conjugated streptavidin to amplify the signal. Beware, however, that you will probably also detect endogenously biotinylated proteins from the plant tissue, so you may need to block the membrane with avidin/biotin as well as the 5% milk before doing the antibody washes.

I've used the streptavidin/HRP with bacterial proteins, and it's worked fairly well.

[Prep!]

than4, on May 18 2010, 06:23 AM, said:

Not sure about the low expression problem, but to detect a protein 15kDa in size I would be running a 15% gel.  I used to look for cytochrome c which was about the same size and always ran a 15%.  Your transfer time seems okay so as not to be transferring the small proteins through the membrane.  Could you try to concentrate your protein by doing an IP from the plant cells?

i agree use a higher percentage of gel and also use a 0.2 micron membrane so tat u dont lose out on the protein!!!
have u ever stained the transferred gel? what is your transfer buffeR>??!!

[KXHK]

Kansas313, on May 17 2010, 11:51 PM, said:

Hi All,

I need help in detecting a small protein (~15kD) with western blot. I believe that this protein has very low expression in plant tissues. I am able to detect the positive control (50 ng expressed protein) but not from the plant tissues. I load lot of protein on the 12% Bis-Tris gel (160 ug/well) from the plant tissues and transfer for 1.30 Hr at 21 V. Do blocking with 5% milk in TBST for 1 Hr and primary antibody incubation in 1 % milk at 4c overnight.

Please help me with any suggestions or protocol for my problem.

Thanks,

Try to load more protein content, use higher concentration of primary antibody (e.g. 1:200) or longer incubation time of primary antibody (i.e. R.T. for 3hr plus 4C overnight).