protein complex purification, western shows tag protein was pulled out, but silv - (Jan/24/2007 )
as title. I try to purify the protein complex by tagging my protein with FLAG tag. I did the purification and from western blot I saw the band of my protein very strong in the elution and no band in control cells(not transfected with my protein) . but silver stain shows no difference between the two different cell lines. and i can not tell my protein in silver stain even I saw hugh band in western. i need to go to mass spectrum to identify the complex. but now even the purification seems working from the western. but if no difference beween in silver stain, i have no idea what is going on. could anyone give me some advices. thanks
-cathy-
anyone has similar experience? thanks a lot
-cathy-
huge band doesn't necessarily mean that you can see the protein also in silverstaining. WB can be very selective and depending on the staining procedure/time give a very huge signal with very little protein. in the silverstaining you see all proteins, if your protein is e.g. less than 1% of whole protein, you will hardly see it.
-Kersten-
Yes..
More protein, more protein! When you get more protein, you may see the difference between positive and control.
-yeping-
QUOTE (cathy @ Jan 24 2007, 04:33 PM)
as title. I try to purify the protein complex by tagging my protein with FLAG tag. I did the purification and from western blot I saw the band of my protein very strong in the elution and no band in control cells(not transfected with my protein) . but silver stain shows no difference between the two different cell lines. and i can not tell my protein in silver stain even I saw hugh band in western. i need to go to mass spectrum to identify the complex. but now even the purification seems working from the western. but if no difference beween in silver stain, i have no idea what is going on. could anyone give me some advices. thanks
abundancy polypeptides detected by silver staining are numerous and difficult to assign to a corresponding immunosignal; better use 2D gel electrophoresis for better assessment
-The Bearer-
QUOTE (The Bearer @ Jan 26 2007, 02:15 AM)
QUOTE (cathy @ Jan 24 2007, 04:33 PM)
as title. I try to purify the protein complex by tagging my protein with FLAG tag. I did the purification and from western blot I saw the band of my protein very strong in the elution and no band in control cells(not transfected with my protein) . but silver stain shows no difference between the two different cell lines. and i can not tell my protein in silver stain even I saw hugh band in western. i need to go to mass spectrum to identify the complex. but now even the purification seems working from the western. but if no difference beween in silver stain, i have no idea what is going on. could anyone give me some advices. thanks
abundancy polypeptides detected by silver staining are numerous and difficult to assign to a corresponding immunosignal; better use 2D gel electrophoresis for better assessment
I TCA precipite my elution and load on my gel again. lots of extra protein come out and still no difference between my control and transfected cells.
Do you think could be too much abundance proteins covers my protein? if so, how could i get rid of most of the abundant proteins?
-cathy-
QUOTE (yeping @ Jan 25 2007, 06:09 AM)
Yes..
More protein, more protein! When you get more protein, you may see the difference between positive and control.
More protein, more protein! When you get more protein, you may see the difference between positive and control.
IF I use more proteins, then should have more abundant proteins which could still cover my proteins. what do you think
-cathy-
QUOTE (cathy @ Jan 26 2007, 12:45 PM)
QUOTE (yeping @ Jan 25 2007, 06:09 AM)
Yes..
More protein, more protein! When you get more protein, you may see the difference between positive and control.
More protein, more protein! When you get more protein, you may see the difference between positive and control.
IF I use more proteins, then should have more abundant proteins which could still cover my proteins. what do you think
I forget to mention that silver staining is not useful to quantify polypeptides which means differences in amounts are not clearly to distinguish; if you use more protein try to stain with CBB
-The Bearer-
QUOTE (The Bearer @ Jan 26 2007, 01:21 PM)
QUOTE (cathy @ Jan 26 2007, 12:45 PM)
QUOTE (yeping @ Jan 25 2007, 06:09 AM)
Yes..
More protein, more protein! When you get more protein, you may see the difference between positive and control.
More protein, more protein! When you get more protein, you may see the difference between positive and control.
IF I use more proteins, then should have more abundant proteins which could still cover my proteins. what do you think
I forget to mention that silver staining is not useful to quantify polypeptides which means differences in amounts are not clearly to distinguish; if you use more protein try to stain with CBB
I use 6mg protein. about CBB stain, I will try. But in quite lot of papers, they use silver stain and found the one transfected got lots extra bands compared to untransfected. I am bit confused
-cathy-
QUOTE (cathy @ Jan 31 2007, 02:17 PM)
QUOTE (The Bearer @ Jan 26 2007, 01:21 PM)
QUOTE (cathy @ Jan 26 2007, 12:45 PM)
QUOTE (yeping @ Jan 25 2007, 06:09 AM)
Yes..
More protein, more protein! When you get more protein, you may see the difference between positive and control.
More protein, more protein! When you get more protein, you may see the difference between positive and control.
IF I use more proteins, then should have more abundant proteins which could still cover my proteins. what do you think
I forget to mention that silver staining is not useful to quantify polypeptides which means differences in amounts are not clearly to distinguish; if you use more protein try to stain with CBB
I use 6mg protein. about CBB stain, I will try. But in quite lot of papers, they use silver stain and found the one transfected got lots extra bands compared to untransfected. I am bit confused
silver is sensitive in the low nanomolar range dtecting more band s than the more insensitive CBB but silver is not useful for quantification as there is a steep slope of sensitivit; CBB has a more even slope of sensitivity
-The Bearer-