Hi,

Your western protocol is fine.

Compare the sequencing result using expasy translate tool to make sure the reading frame is not altered after knock out. 

If you have the antibody binding amino acid sequence of the protein, make sure its not at the site of knock out performed.

 Did you use a molecular weight marker along with it and ensure that the protein entered the gel? If so, you may try a higher concentration of primary antibody (1:100). 

I could not find any other problems with your conditions. Since it is not working with western, if the primary antibody is compatible, I would suggest doing immune fluorescence for your normal and knockout cells.

I hope this helps. Goodluck

Are the antibodies directed against the native or the denatured protein (structural or sequence motif)? Many antibodies directed against native proteins don't work in blots, but are fine for immune precipitation or histochemistry. The spec sheet that came with the antibody should tell you that.

Another issue is the use of 8% PAGE gels for a 250 kDa protein. The protein wouldn't enter very far into such a gel, and it may be difficult to get out again during blotting. I'd try a much lower %T (say, a gradient from 3-10%), and stabilise the resulting gel with 0.5% agarose (which becomes restrictive only for particles of many MDa).

At least in my hands, tank blotters work much better for large proteins than semidry, and instead of NC I always us PVDF membranes (more complete binding of proteins, no tendency to break during incubations). Dunn's transfer buffer (10 mM NaHCO3, 3 mM Na2CO3) is more reliable than Towbin's and also a lot cheaper. In my hands, it works even better with 100 µM SDS (no methanol!).

If question says, knock out of T-type channel isoform leads to its inactivation by inhibiting its protein expression. In western we detect no band(knock out) versus band in control(intact sample). If you can not see the band in the Control also increase the protein quantity (and use little more stacking gel 1.5cm). As I said your westren conditions did work with me for mTOR(with SDS and no methanol in transfer buffer).

Dear All,

Thank you very much for all your suggestions. I'll try all the parameters suggested by you all and get back to you.

Thank you  once again 

Best regards

Sudha

This protocol from Nature is designed for big/small proteins. Hope it helps. 

Dear all,

Based on your suggestions, i did western blot for high molecular weight protein. i have separated the protein in 8% Tris-bis PAGE gel and transfer at constant voltage of 35V for 3 hours under cold condition. The blot was blocked in 5% non-fat milk in TBST (0.1% Tween 20) and blots were incubated overnight in primary antibody and the next day the blot was washed thrice 10 mins each in TBST and incubated in secondary antibody for an hour. The blot was washed again thrice for 10mins each and ECL substrate (1:1) was used and developed in X ray.

For the target protein;

Primary antibody is polyclonal rabbit

secondary antibody is goat anti-rabbit-HRP.

Loading control:

b actin polyclonal rabbit

secondary antibody is goat anti-rabbit-HRP

Herewith i have attached the images of the result i got. Img_1496 is for the target protein and img_1495 is for the loading control. As you see in the first image.

The top one in the Img_1496, i could see the desire band but in addition to that i have seen some vertical streaks below the band.

The bottom one in the Img_1496 is worse, its completely black. I have used the same condition for the bottom one as of top one. Both the targets are around 250-270KD size. I'm not sure why this black streaks and black background is observed in the blot.

Ironically, the loading control of the respective blots as seen in Img_1495 doesn't show any vertical streaking or black background. The secondary antibody for loading control and target protein are same. But still, i can't get the answer why i'm getting this result. Could you please help me with this.

Thanks

Best regards

Sudha

Hello sudha,

Your western protocol is fine.

- nitrocellulose membrane activation by methanol ?

- You can try, gel and transferred to nitrocellulose membrane of 100V for 1 hours, in cold condition.

- if didn't work.

- Then knock-out method ??? (doubt)

knock out of T-type channel isoform leads to its inactive or inhibiting its protein expression. In western will not detect the band (knock out)and what about control????