Trouble with equal loading - (Dec/21/2011 )
Hi everybody,
it may sound weird but I could not optimize the proper protein concentration measurement for six month.
I have tried out several methods (Pierce Bradford, Biorad both microplate and cuvette, QUBIT) but with all these methods I came up with nonequal loading. There should be something interacting with these reagents in my Lysis Buffer. (NP40 1% and freshly add protease inhibitor cocktail of Roche, PMSF and DTT). I even can not draw proper standart curve, the value are not proportional.
Does anyone help me with this problem? I would appreciate any ideas and suggestions....
best
How long you boiling your sample, and don't forget to spin the sample after boiling it, before you go for measuring the protein concentration.
Incomplete boiling often leads incorrect protein measurement and ends in unequal loading.
What was range you using to make your protein standard curve
I don't boil my samples prior to measuring total protein content, I boil it just before loading it onto my SDS-PAGE.
I'm happy with the consistency of the Bio-Rad BCA kit (reagents A and B ). We use BSA as the standard protein.
science noob on Thu Dec 22 09:59:44 2011 said:
I don't boil my samples prior to measuring total protein content, I boil it just before loading it onto my SDS-PAGE.
I'm happy with the consistency of the Bio-Rad BCA kit (reagents A and B ). We use BSA as the standard protein.
What is the logic in boiling the sample before loading not before measuring the protein.
In non boiled sample proteins interact with other proteins, DNA all possible thing the sample and if you measure such sample, all proteins are not available for protein measurement and you end with unequal loading. So boiling sample before measuring is advisable.
Biouday on Thu Dec 22 11:44:40 2011 said:
science noob on Thu Dec 22 09:59:44 2011 said:
I don't boil my samples prior to measuring total protein content, I boil it just before loading it onto my SDS-PAGE.
I'm happy with the consistency of the Bio-Rad BCA kit (reagents A and B ). We use BSA as the standard protein.
What is the logic in boiling the sample before loading not before measuring the protein.
In non boiled sample proteins interact with other proteins, DNA all possible thing the sample and if you measure such sample, all proteins are not available for protein measurement and you end with unequal loading. So boiling sample before measuring is advisable.
not so. you want to know the protein concentration of the lysate so that you can use equal amounts of protein in your samples. measuring protein after boiling in sample buffer can be problematic because of the presence of relatively high concentrations of sds, bme and, especially, bromphenol blue (can be left out until just before loading but will interfere with readings if not omitted).
UmiYasi on Wed Dec 21 21:44:01 2011 said:
Hi everybody,
it may sound weird but I could not optimize the proper protein concentration measurement for six month.
I have tried out several methods (Pierce Bradford, Biorad both microplate and cuvette, QUBIT) but with all these methods I came up with nonequal loading. There should be something interacting with these reagents in my Lysis Buffer. (NP40 1% and freshly add protease inhibitor cocktail of Roche, PMSF and DTT). I even can not draw proper standard curve, the value are not proportional.
Does anyone help me with this problem? I would appreciate any ideas and suggestions....
best
all of the additives to the lysis buffer that you mention will affect the protein determination. you may be able to account for buffer effects with proper, stringent, blanking but not always. you may have to dilute or dialyze the sample to reduce the concentration of these additives to a workable level.
It would be useful to know what values for the standard curve you are using. If it's too high or too low, the assay is useless. In addition, if your sample absorbances are too high, you'll get the same problem.
How are you gauging equal protein load in your western? Certain conditions can cause differences to so-called housekeeping genes that might look like a significant difference. For example, if you are looking at signaling, you may end up with a difference in the filamentous to monomeric actin, and since F-actin is less soluble you might throw some of it out with your pellet, depending on how you treat your samples. This would cause perfectly equal protein loads to look bad if you're using actin.
Do the bands have the same width on your blot? If they are different, it could be due to differences in the loaded total volume.
There are quite a few questions to ask about the results. The fact that you can't get a standard curve to work implies some critical error. How old is the reagent you're using? Are you reading at the right absorbances?
deadally on Thu Dec 22 15:40:58 2011 said:
It would be useful to know what values for the standard curve you are using. If it's too high or too low, the assay is useless. In addition, if your sample absorbances are too high, you'll get the same problem.
How are you gauging equal protein load in your western? Certain conditions can cause differences to so-called housekeeping genes that might look like a significant difference. For example, if you are looking at signaling, you may end up with a difference in the filamentous to monomeric actin, and since F-actin is less soluble you might throw some of it out with your pellet, depending on how you treat your samples. This would cause perfectly equal protein loads to look bad if you're using actin.
Do the bands have the same width on your blot? If they are different, it could be due to differences in the loaded total volume.
There are quite a few questions to ask about the results. The fact that you can't get a standard curve to work implies some critical error. How old is the reagent you're using? Are you reading at the right absorbances?
good points about range and inability to make a proper standard curve.
however, the original poster never mentions blotting. s/he only refers to sds-page.
deadally on Thu Dec 22 15:40:58 2011 said:
It would be useful to know what values for the standard curve you are using. If it's too high or too low, the assay is useless. In addition, if your sample absorbances are too high, you'll get the same problem.
How are you gauging equal protein load in your western? Certain conditions can cause differences to so-called housekeeping genes that might look like a significant difference. For example, if you are looking at signaling, you may end up with a difference in the filamentous to monomeric actin, and since F-actin is less soluble you might throw some of it out with your pellet, depending on how you treat your samples. This would cause perfectly equal protein loads to look bad if you're using actin.
Do the bands have the same width on your blot? If they are different, it could be due to differences in the loaded total volume.
There are quite a few questions to ask about the results. The fact that you can't get a standard curve to work implies some critical error. How old is the reagent you're using? Are you reading at the right absorbances?
Firstly, thank you all for your reply.
I am doing western for cycle cycle expression of certain protein, so equal loading is very important for me. I need to denect even 30% increase in protein expression. And the loading control I am using is known to be same throughout the cell cycle.
Concerning my standard curve, I am getting negative value till 500ug/ul BSA. Higher concentrations come up with improper increase. I think this is mostly due to the contents of my Lysis buffer. 1.5 mg/ml BSA standard gives higher OD than 2mg/ml; for all other standard values I dilute my stock BSA (2mg/ml) with my Lysis Buffer).
About boiling the lysate before concentration measurement, I never tried it, I just boil and spin just before loading to gel.
After running, I do semidry transfer (also tested if I have a problem with transfer system by loading same amount of same samples, seems to work good). Use homemade ECL and finally develop the film manually.
My reagents( both the kit and my lysis buffer) are fresh and I am following the protocol of the kit, meaning measuring at 595 nm.
I neglect the loading if there is a difference in width of my bands, but I encounter this problem even with same width of the bands.
I will try to dilute my lysis buffer and samples before measuring, if there anything is interfering...
best regards..
Make sure you are using a 4 parameter equation for the standard curve - it is NOT a straight line!.