western blot problem - no expression on stable transfection. (Jun/03/2008 )
For last couple of months i have been creating a stable transfectio of the transcription al fcator, I selected my cells with G418 over 4 week s and still doing so , i started with 400mg/ml all way up to 800mg/ml. Upon inspection i saswe clones and then split cells and let them gro w a bit more, now i need to analyze the expression of targeted gene via western. My gene has HA tag so i use HA an tiboty for western So here is the problem, muy transient transfection i can see the expresion of the all my clones but wneh i check the stable transfected ones i dont see any expression, and i am wodering if something is worng with western or i just dont have expression on those stable cell lines. please help cause i dont know what to do. If there is no stable trasnfection whta make the cells survive the under the G418. 
-mama wa nyumbani-
QUOTE (mama wa nyumbani @ Jun 3 2008, 10:38 AM)
For last couple of months i have been creating a stable transfectio of the transcription al fcator, I selected my cells with G418 over 4 week s and still doing so , i started with 400mg/ml all way up to 800mg/ml. Upon inspection i saswe clones and then split cells and let them gro w a bit more, now i need to analyze the expression of targeted gene via western. My gene has HA tag so i use HA an tiboty for western So here is the problem, muy transient transfection i can see the expresion of the all my clones but wneh i check the stable transfected ones i dont see any expression, and i am wodering if something is worng with western or i just dont have expression on those stable cell lines. please help cause i dont know what to do. If there is no stable trasnfection whta make the cells survive the under the G418.
I don't think it's because of the western procedure, it must be the transfection. and please pay more attention to your English writing, you have many errors.
-Curtis-
Is the stable transfection under an inducible system? It may be that you need to add something (tetracycline is a common one for inducible systems) to get the protein to express if the system is an inducible one.
-bob1-
QUOTE (mama wa nyumbani @ Jun 3 2008, 09:38 AM)
For last couple of months i have been creating a stable transfectio of the transcription al fcator, I selected my cells with G418 over 4 week s and still doing so , i started with 400mg/ml all way up to 800mg/ml. Upon inspection i saswe clones and then split cells and let them gro w a bit more, now i need to analyze the expression of targeted gene via western. My gene has HA tag so i use HA an tiboty for western So here is the problem, muy transient transfection i can see the expresion of the all my clones but wneh i check the stable transfected ones i dont see any expression, and i am wodering if something is worng with western or i just dont have expression on those stable cell lines. please help cause i dont know what to do. If there is no stable trasnfection whta make the cells survive the under the G418.
1. Are these adherent cells? If so, you need to select clones, just collecting pools may not give you sufficient number of stably expressing clones.
2. Use your transient transfection lysate (at 2 or 3 dilutions) as control on the same western, that would tell you if there was anything wrong with western.
3. Stablly expressing cells have much lower expression as compared to transient, so if your western sensitivity is no good, you will not detect stable expression.
4. And yes, while your cells are happily growing in G418, it may be worth reading engilsh novels to improve english
-cellcounter-
QUOTE (cellcounter @ Jun 3 2008, 09:44 PM)
QUOTE (mama wa nyumbani @ Jun 3 2008, 09:38 AM)
For last couple of months i have been creating a stable transfectio of the transcription al fcator, I selected my cells with G418 over 4 week s and still doing so , i started with 400mg/ml all way up to 800mg/ml. Upon inspection i saswe clones and then split cells and let them gro w a bit more, now i need to analyze the expression of targeted gene via western. My gene has HA tag so i use HA an tiboty for western So here is the problem, muy transient transfection i can see the expresion of the all my clones but wneh i check the stable transfected ones i dont see any expression, and i am wodering if something is worng with western or i just dont have expression on those stable cell lines. please help cause i dont know what to do. If there is no stable trasnfection whta make the cells survive the under the G418.
1. Are these adherent cells? If so, you need to select clones, just collecting pools may not give you sufficient number of stably expressing clones.
2. Use your transient transfection lysate (at 2 or 3 dilutions) as control on the same western, that would tell you if there was anything wrong with western.
3. Stablly expressing cells have much lower expression as compared to transient, so if your western sensitivity is no good, you will not detect stable expression.
4. And yes, while your cells are happily growing in G418, it may be worth reading engilsh novels to improve english
1. yes, they are adherent cells, and my boss is not interested on the individual clones, with that im stuck with the mix population.
2. I have done transient transfection with 293 cells with no problem so, so i know my vector express my gene. i dont know what you mean when you say 2 to 3 dilution.
3. antibody that i use for western is anti-HA so i know its pretty good, which cover the sensitivity.
4 darn, sorry for the english i know its not up to par but i think i was typing to fast.
-mama wa nyumbani-
QUOTE (bob1 @ Jun 3 2008, 06:06 PM)
Is the stable transfection under an inducible system? It may be that you need to add something (tetracycline is a common one for inducible systems) to get the protein to express if the system is an inducible one.
No, its not under inducible systeams.
-mama wa nyumbani-
QUOTE (Curtis @ Jun 3 2008, 04:57 PM)
QUOTE (mama wa nyumbani @ Jun 3 2008, 10:38 AM)
For last couple of months i have been creating a stable transfectio of the transcription al fcator, I selected my cells with G418 over 4 week s and still doing so , i started with 400mg/ml all way up to 800mg/ml. Upon inspection i saswe clones and then split cells and let them gro w a bit more, now i need to analyze the expression of targeted gene via western. My gene has HA tag so i use HA an tiboty for western So here is the problem, muy transient transfection i can see the expresion of the all my clones but wneh i check the stable transfected ones i dont see any expression, and i am wodering if something is worng with western or i just dont have expression on those stable cell lines. please help cause i dont know what to do. If there is no stable trasnfection whta make the cells survive the under the G418.
I don't think it's because of the western procedure, it must be the transfection. and please pay more attention to your English writing, you have many errors.
so, should i assume that SCLC cells are hard to transfect compare to hek293( which i usually, use for transient transfection), because my transient transfection works fine.Another trouble some issue is that, i know when the cells have not been transfected an i add G418 they all die because it happen for one of the clone.So, i think that either the gene stop expressing.???????? Thanks for suggestion and i think i was typing very fast when i was posting( even know my english still need some improvement)
-mama wa nyumbani-
QUOTE (mama wa nyumbani @ Jun 6 2008, 07:38 AM)
1. yes, they are adherent cells, and my boss is not interested on the individual clones, with that im stuck with the mix population.
You may still make individual clones to see if you get higher expression with some clones.
2. I have done transient transfection with 293 cells with no problem so, so i know my vector express my gene. i dont know what you mean when you say 2 to 3 dilution.
Because stable expression is much less, if you compare transient with stable, you may feel you have no stable expression. So, dilute the transient transfection to say 1:5, 1:10, 1:25, run on the same gel, do western, and still you detect a band there, but not in your stable, you can move on to some other projects or bosses.
3. antibody that i use for western is anti-HA so i know its pretty good, which cover the sensitivity.
It is not only ab, it is also technique, so having a diluted transient control there is always good.
4 darn, sorry for the english i know its not up to par but i think i was typing to fast.
Darn
You may still make individual clones to see if you get higher expression with some clones.
2. I have done transient transfection with 293 cells with no problem so, so i know my vector express my gene. i dont know what you mean when you say 2 to 3 dilution.
Because stable expression is much less, if you compare transient with stable, you may feel you have no stable expression. So, dilute the transient transfection to say 1:5, 1:10, 1:25, run on the same gel, do western, and still you detect a band there, but not in your stable, you can move on to some other projects or bosses.
3. antibody that i use for western is anti-HA so i know its pretty good, which cover the sensitivity.
It is not only ab, it is also technique, so having a diluted transient control there is always good.
4 darn, sorry for the english i know its not up to par but i think i was typing to fast.
Darn
-cellcounter-
QUOTE (cellcounter @ Jun 6 2008, 07:50 AM)
QUOTE (mama wa nyumbani @ Jun 6 2008, 07:38 AM)
1. yes, they are adherent cells, and my boss is not interested on the individual clones, with that im stuck with the mix population.
You may still make individual clones to see if you get higher expression with some clones.
2. I have done transient transfection with 293 cells with no problem so, so i know my vector express my gene. i dont know what you mean when you say 2 to 3 dilution.
Because stable expression is much less, if you compare transient with stable, you may feel you have no stable expression. So, dilute the transient transfection to say 1:5, 1:10, 1:25, run on the same gel, do western, and still you detect a band there, but not in your stable, you can move on to some other projects or bosses.
3. antibody that i use for western is anti-HA so i know its pretty good, which cover the sensitivity.
It is not only ab, it is also technique, so having a diluted transient control there is always good.
4 darn, sorry for the english i know its not up to par but i think i was typing to fast.
Darn
You may still make individual clones to see if you get higher expression with some clones.
2. I have done transient transfection with 293 cells with no problem so, so i know my vector express my gene. i dont know what you mean when you say 2 to 3 dilution.
Because stable expression is much less, if you compare transient with stable, you may feel you have no stable expression. So, dilute the transient transfection to say 1:5, 1:10, 1:25, run on the same gel, do western, and still you detect a band there, but not in your stable, you can move on to some other projects or bosses.
3. antibody that i use for western is anti-HA so i know its pretty good, which cover the sensitivity.
It is not only ab, it is also technique, so having a diluted transient control there is always good.
4 darn, sorry for the english i know its not up to par but i think i was typing to fast.
Darn
thanks, i will try ur suggestion and keep you posted( on western with diluted transient plus stable)
ps how do you isolate the individual clones
-mama wa nyumbani-
QUOTE (mama wa nyumbani @ Jun 6 2008, 09:34 AM)
ps how do you isolate the individual clones
By using cloning rings: For protocols see the link below..
http://search.vadlo.com/b/q?sn=158621799&a...ng%22&rel=0
...
-cellcounter-