イムノブロット解析

hello,

for extraction of proteins from cell if we want to lyse whole cell we should use antiprotease .Could we use only PMSF for this purpose or we must use also other antiprotease  with it...if our desired protein is chemokine that secretes to supernatant of our cell culture flask how could we extract it ....thanks alot for your kinds...

It would be great if people submit discussing the troubleshooting of western blots (one of such kind has been dealt in this video..air bubbles trapped in western blots..coz the blotting membrane was put very rapidly in to the transfer buffer for pre wetting)

Post them!

Sudheendra.

Is there a video for co-immunoprecipitation???

How do you quantify the blots with Image-J

i try to do dot-blot but after using lysis buffer when i lay 1 microlitter of it on PVDF membrane pvdf was lysed what should i do?

I didn't see you wash after the milk blocking. You used the antibody right after. Is this how you really do it, or did I miss a step?

Couple questions

Did you do a wash after the milk blocking? or did I miss something?

I am tring to download the pdf..for somereason it is saving me in a very strange format that my pc doesn't recognize.

Can u explain?

THanks

WIL

You do not have to wash after blocking.

You can block then put in primary overnight. After primary and after secondary is where you have to wash so you dont have a lot of background. Works all the time for me...

Dr. Gallagher or Dr. Chakavarti! Could I contact you by E-mail directly, please?

Please contact me at seang@uvp.com

Best regards,

Sean Gallagher

in which step can we leave the system overnight else?

1. At the end of procedure when we dye the Immobilon-P membrane with BioRad 1immu-Star^{TM AP}  Chemo luminescent Protein Detection System: Do we have to remove excess of dye from membrane just before luminescent detection?