Lots of bands on immunoblot for an expressed protein - (Nov/28/2006 )
Hey guys,
I got some new plasmids and have been using them for transfection into Cos-7 cells and subsequently preparing lysates and IPs from them to run on a gel and transfer to a blot. The first time my blot came out with tons of bands especially in the lysates. I've run 3 different gels with pretty much the same results, sometimes less bands but still far too many.
So, I ran a known set of IPs and Lysates. Same thing. Lots of bands where I know there shouldn't be any because this is an experiment I've done before with clean results on the blot.
The known IP's and Lysates were already prepared and had been used before with clean results, so I doubt its my preparation technique.
I've checked and even had someone look over my shoulder as I load the gel and had no complaints. Neither samples ever come off ice for more time than it takes to load them. I am at a loss for why I'm getting so much artifact. Anyone encounter this problem before? How did you correct for it? Thanks in advance.
Amelia
try asking here http://www.protocol-online.org/forums/inde...showtopic=21665
it concerns loading dyes and questions about blots...
Have you recently changed your antibody batch by any chance? If so, you might need to re-titrate it, as there are huge differences in the way an antibody works from batch to batch...
I got some new plasmids and have been using them for transfection into Cos-7 cells and subsequently preparing lysates and IPs from them to run on a gel and transfer to a blot. The first time my blot came out with tons of bands especially in the lysates. I've run 3 different gels with pretty much the same results, sometimes less bands but still far too many.
So, I ran a known set of IPs and Lysates. Same thing. Lots of bands where I know there shouldn't be any because this is an experiment I've done before with clean results on the blot.
The known IP's and Lysates were already prepared and had been used before with clean results, so I doubt its my preparation technique.
I've checked and even had someone look over my shoulder as I load the gel and had no complaints. Neither samples ever come off ice for more time than it takes to load them. I am at a loss for why I'm getting so much artifact. Anyone encounter this problem before? How did you correct for it? Thanks in advance.
Amelia
may be I missed the point but it is normal that lysates contain thousands of proteins, and IP enriches your protein of interest but there may be still many diiferent proteins which are co-precipitated. So, I do not know how a "clean" result should look like...
may be I missed the point but it is normal that lysates contain thousands of proteins, and IP enriches your protein of interest but there may be still many diiferent proteins which are co-precipitated. So, I do not know how a "clean" result should look like...
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I could very well be the one confused here too 
I mention the lysates in particular because I am getting so many bands it is hard to determine what is real from what is artifact. However, the same occurs in my IP blot as well just to a smaller degree making it more readable but with the lysates coming out the way they are I have a hard time comparing the two.
I suppose in using the word clean I am referring to not only the amount of bands but the intensity with which they highlight on the blot. The best example I can provide is I have an experiment we've run many times for confirmation and the blots, both IPs and lysates, have clear decisive bands. Running the same thing again all the way from transfection to blot development I'm getting literally hundreds of bands in each lane. Far more than I've ever gotten from this experiment. If I can get my hands on the scanner next door I'll upload some comparison pics. Maybe that'll clarify what I mean, cause I'm banging my head against the wall trying to determine the cause of this.
to follow the facts - how the blots are stained? Ponceau S, amido black, CBB, india ink, other otein stains? or are you talking about immunoblots?
Immunoblots, sorry. Showing my green edges. Its a Western Blot that I block overnight with BSA and then am using either a GFP primary antibody or 9E10 primary with a horseradish periooxidase 2dary. The GFP antibody is a new batch so I could accept that it might be the culprit except that my 9E10 is a tried and true batch.
It has been suggested that my proteins are degrading somehow but I don't know where in the process that would be occuring.
Hey guys,
I got some new plasmids and have been using them for transfection into Cos-7 cells and subsequently preparing lysates and IPs from them to run on a gel and transfer to a blot. The first time my blot came out with tons of bands especially in the lysates. I've run 3 different gels with pretty much the same results, sometimes less bands but still far too many.
So, I ran a known set of IPs and Lysates. Same thing. Lots of bands where I know there shouldn't be any because this is an experiment I've done before with clean results on the blot.
The known IP's and Lysates were already prepared and had been used before with clean results, so I doubt its my preparation technique.
I've checked and even had someone look over my shoulder as I load the gel and had no complaints. Neither samples ever come off ice for more time than it takes to load them. I am at a loss for why I'm getting so much artifact. Anyone encounter this problem before? How did you correct for it? Thanks in advance.
Amelia
may be I missed the point but it is normal that lysates contain thousands of proteins, and IP enriches your protein of interest but there may be still many diiferent proteins which are co-precipitated. So, I do not know how a "clean" result should look like...
yes lysate contains thousands of protein but when we are doing IP and using specific antibody it should be only one band at the end, i am doing cell lysate IP also if u want to recheck ur protocol with me its fine.
another thing i am sorry are u using protease inhibitors?
another thing i am sorry are u using protease inhibitors?
I get all sort of bands in both my lysates and my IPs with no explanation.
Here is what I typically do:
After cell transfection is complete I lyse them in:
Modified RIPA buffer, with fresh protease inhibitors and PMSF. Incubate 1ml to each plate on ice for 10-15min. Scrap them off into an eppendorf tube, spin them down ~16000xg for 10min. Decant the supernatant and discard the pellet.
From the supernatant I take 100ul for lysate prep, add 50ul 3X Sample Buffer, boil for 5 min, cool them down and either store them at -20C or use them in a gel, still they go into -20C for storage after.
For IP's:
I use the remaining supernatant and add 5ul of my chosen antibody. I incubate this in our cold room overnight on a rocker.
The next day bring them on ice to the lab and add 50ul of Protein G Sepharose beads and back to the coldroom for 1.5-2 hrs rocking away.
Then I spin them down, aspirate off the fluid and wash x3 in modified RIPA buffer with a spin ~30sec between each wash.
After the 3rd wash I ensure all the fluid is adaquately removed without disturbing my beads, add 70ul 3X Sample Buffer, boil 5min and cool down for use or storage in -20C.
For the gel and transfer:
I pour my gel according to the % I need, pour the stacking gel once its all set I wash out my wells with a little Running Buffer and leave enough for loading my samples.
Typically I can load between 10-20ul depending on which comb I've used for my gel. I try very hard not overload them to prevent leakage into the next well.
Once loaded I fillup my chambers with Running Buffer and let is go at 125V for about 45min - 1hr. I don't run it all the way to the bottom of the gel a little over 3/4.
Once complete I prep my PVDF membrane, wash in methanol, rinse in diH2O and let it hang out in 1X Transfer Buffer with 20% Methanol til I'm ready to put it on the gel.
I soak my pads and filter paper in the same buffer and start making my sandwich.
2 pads, 1 filter paper, Gel, Membrane, Filter paper, 3 pads.
I toss it in the box fill up the chambers with Transfer Buffer and let it run a minimum of 1.5hrs sometimes up to 3 hrs.
After the run I remove the membrane give it a quick run through some 1X PBS and into
1X PBS/Tween-20 0.1% w/ 1% BSA solution overnight in the cold room.
Next day 1hr primary ab usually 1:2000 dilution
PBS/Tween wash 15 min
1hr secondary ab between 1:5000 and 1:10000 dilutions
3, 15min washes in PBS/Tween
Let set in 1X PBS while I prep the ECL
And still all I get are non-specific crazy, wild bands!
This is from memory with quick references to my available protocols. I think it best reflects my flow.