I have change all the buffer and also replaced aps and acrylamide still not getting good resolution of bands and gels is smeared from the bottom.My protein size is 23 kd. Kindly provide your valuable suggestions.
Hello Sidra Ayub
There could be several reasons why your gel is not resolving properly. Can you specify what % of acrylamide you are using? If you carefully notice, I don't think it's the gel that is causing the problem because your Molecular marker is clearly getting resolved properly. In my opinion, the problem lies with your samples. Either they have a high concentration of salt or any denaturant, or the protein concentration you're using is too high (Gel 2/3), or the pH of your samples is not correct. I can see in Gel 3, that the middle lane has got a high concentration of protein (relative to others), and also it shows a yellow coloration at the bottom. This yellow color may be because your sample's pH is acidic and not according to what it should be (that of the loading dye). Do your samples change the color of the loading dye during preparation, if so, try adding a drop of 1N NaOH to them to neutralize the pH back to the original.
Hopefully, this helps you in figuring out the underlying problem. Good luck!
Hello @Jatin Chadha
I am using 30 percent of acrylamide.
I am tring to extract proteins from Chlydomonas Reinhardtti ,which is alage and gives yellowish and greenish colour to the sample. Yes, in gel 2 I have used a higher concentration sample but whats wrongs with other samples they are too not resolved properly and are smeared.
Thanks for your insightful response
30% acrylamide is your stock. From that, you add a certain amount for preparing your gel. What is the % of acrylamide gel are you using? 12%, 15%?
What is the composition of your lysis buffer? I think it probably has too much salt(s) in it.
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