Hi, I am trying to detect cMyc with W.B
I used iBlot semi dry system and Ponceau was quite good. I blocked with 5% Milk and incubated overnight with primar ab (4°). The day after I washed with PBS-T 0.01% and incubated for 1h with secondar ab. After 3 washes (10; 5; 5 min) I detected with select ECL. The imaging was obtained with chemidoc. Since at 2 seconds of exposure the signal was not so good I decided to increase time of exposure but the membrane became black. Do you have any idea how to avoid it?
Here you can find the picture of it
Thanks
Hi Blerta, there are many ways to improve the signal in WB but it relys much upon the specific antibody. You should check the manufacturer's instructions to see if changing the blocking and dilution conditions to BSA instead of milk might improve your signal to noise ratio, but besides, you can try to dliute the primary antibody; sometimes working with too concentrated solutions is worse. I would use a series of conditions, a small screening test, cehcking milk/BSA, 1/1000 - 1/10000 dilutions, and finally trying to play with the imaging software or the ECL solution to try to obtain a good signal. I've never used an antibody against cMyc so the shortest way would probably trying to find somebody who uses it in WB and can tell you some tricks about reading the blots and conditions of use for the antibody. Sorry if this wasn't much help!
Hi Blerta, there are many ways to improve the signal in WB but it relys much upon the specific antibody. You should check the manufacturer's instructions to see if changing the blocking and dilution conditions to BSA instead of milk might improve your signal to noise ratio, but besides, you can try to dliute the primary antibody; sometimes working with too concentrated solutions is worse. I would use a series of conditions, a small screening test, cehcking milk/BSA, 1/1000 - 1/10000 dilutions, and finally trying to play with the imaging software or the ECL solution to try to obtain a good signal. I've never used an antibody against cMyc so the shortest way would probably trying to find somebody who uses it in WB and can tell you some tricks about reading the blots and conditions of use for the antibody. Sorry if this wasn't much help!
Hi Bletra, every step you did is correct but while adding substrate for membrane better to keep the membrane inside the chemi doc and then add the substrate and incubate their only and thus it increases the senstivity of signal and better to check the dilution pattern of your 2' antibody according to manufacturers instruction and sometimes low dilution and improper blocking also may give such images , good luck ! thank you
Hello Blerta
I have worked with several antibodies in western blot analysis and I have had this trouble sometime. Although I have not worked with cMyc, I have resulted this by a block with 5% Milk for 6 hours at TAº and an incubation with diluite primary antibody at 4ºC over night. The dilutions that you can use is according to manufacturer's instructions, for example in high , intemedia and low dilution. Check whether others workmates have the same trouble. In addition, you could consider changing by other source of milk.
Also you can try compared different substrate to get the posibility of different sensivity of signal for your protein.
Best regards
Thanks to all of you for your answers. Beyond your suggestions what I did was to change the secondar Ab (by using one from Santa cruz instead the previous one from Sigma) and several washes of 20min each. The result was much better.
Thanks again. Hope my experience will be useful for the others
Concomitantly you may increase percentage of BSA or decrease antibody concentration, good luck.
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