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Does anyone know why there is a partial smear in this SDS PAGE?...
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+ Sds-page
+ Protein analysis
+ Gel electrophoresis
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David J. Hughes
Does anyone know why there is a partial smear in this SDS PAGE?...
Hi Pulu On the contrary, I think ur denaturation is incomplete. Try to do 10 mins at 99 C. Higher acrylamide would also help to distinguish small proteins. Check your SDS buffer in which you make ur protein sample, it might need some extra DTT/b-mercaptoethanol. Often heppens with old buffers, then u add one two drops of them and its perfect.
Hi Pulu On the contrary, I think ur denaturation is incomplete. Try to do 10 mins at 99 C. Higher acrylamide would also help to distinguish small proteins. Check your SDS buffer in which you make ur protein sample, it might need some extra DTT/b-mercaptoethanol. Often heppens with old buffers, then u add one two drops of them and its perfect.
Hi! Pulu, Run not less than a 13-14% gel for the protein your are trying to visualise (16kDa). Also, boil your sample for 9-10 mins before loading and load it immediately after preparing the sample without storing. Run your SDS-PAGE at a constant current of 20mA through out till the dye front reached the bottom of the gel. Moreover, you can probe for your protein and see how it is visualised on western blot..
Hi! Pulu, Run not less than a 13-14% gel for the protein your are trying to visualise (16kDa). Also, boil your sample for 9-10 mins before loading and load it immediately after preparing the sample without storing. Run your SDS-PAGE at a constant current of 20mA through out till the dye front reached the bottom of the gel. Moreover, you can probe for your protein and see how it is visualised on western blot..
Thank you very much for all the suggestions. I have tried to increase the content of acrylamide from 10% to 15%, but it did not work. The smear still existed. At the end, I realized that the smear was caused by the function of the protein itself...
Thank you very much for all the suggestions. I have tried to increase the content of acrylamide from 10% to 15%, but it did not work. The smear still existed. At the end, I realized that the smear was caused by the function of the protein itself...
Hey, I agree with others saying you should use fresh buffers and higher percentage gel. Considering size of your protein, if I were you, I would probably use 20% gel (or at least 15%). Also, it is possible that you have high concentration of wanted protein and maybe you should try higher dilution of your samples (ie. 2x, 3x, not more) together with the one used in this picture and with that 15/20% gel. One more, denaturation (if done with B-merc.ethanol) can be performed for 3-10 minutes on 95-100C, it does not make such a big difference. 7min 100C is very good (at leas in my experience), but I know that some people just leave their samples with b-merc.ethanol overnight at room temperature and denaturation also occurs (I habe not tried this). :) Good luck
PS. Smear bands can also be caused by glycoproteins (the glyco-part).
Hey, I agree with others saying you should use fresh buffers and higher percentage gel. Considering size of your protein, if I were you, I would probably use 20% gel (or at least 15%). Also, it is possible that you have high concentration of wanted protein and maybe you should try higher dilution of your samples (ie. 2x, 3x, not more) together with the one used in this picture and with that 15/20% gel. One more, denaturation (if done with B-merc.ethanol) can be performed for 3-10 minutes on 95-100C, it does not make such a big difference. 7min 100C is very good (at leas in my experience), but I know that some people just leave their samples with b-merc.ethanol overnight at room temperature and denaturation also occurs (I habe not tried this). :) Good luck
PS. Smear bands can also be caused by glycoproteins (the glyco-part).
To Heba Ebeed: Thank you very much for your answer! I did the protein extraction from sample for twice and they showed the same problem. Ok, so do you think the denaturation time is too long (I denaturated for 5 min at 95 C)? I will try your suggestion!
To Jan Piwowarski: Thank you very much for your answer! I will give it a try.
To Heba Ebeed: Thank you very much for your answer! I did the protein extraction from sample for twice and they showed the same problem. Ok, so do you think the denaturation time is too long (I denaturated for 5 min at 95 C)? I will try your suggestion!
To Jan Piwowarski: Thank you very much for your answer! I will give it a try.
I have mainly seen this when my protein is associated with nucleic acids, just as Lokesh said. A quick check might be to determine the A260 nm:A280 nm ratio for nucleic acid contamination of your protein preparation. If nucleic acid is present then try shearing it (Lokesh's suggestion) and/or you could add DNase during lysis.
The other times I've seen odd upwards smears on protein gels when there is no nucleic acid involved is when the protein is very positively charged. They usually aren't so smeary. It could also be that your gel is melting during electrophoresis (similar to Shayne's answer). Was your buffer really hot at the end of your run and are other bands well resolved on your gel?
Note that some proteins are SDS resistant and other proteins aggregate naturally. Maybe if your protein forms SDS-resistant aggregates this might happen (Irfan's answer; i.e. not denatured). There are harsher ways of denaturing protein that are harsher than SDS+boiling. I've heard very low concentrations of TFA+boiling is one of them.
I have mainly seen this when my protein is associated with nucleic acids, just as Lokesh said. A quick check might be to determine the A260 nm:A280 nm ratio for nucleic acid contamination of your protein preparation. If nucleic acid is present then try shearing it (Lokesh's suggestion) and/or you could add DNase during lysis.
The other times I've seen odd upwards smears on protein gels when there is no nucleic acid involved is when the protein is very positively charged. They usually aren't so smeary. It could also be that your gel is melting during electrophoresis (similar to Shayne's answer). Was your buffer really hot at the end of your run and are other bands well resolved on your gel?
Note that some proteins are SDS resistant and other proteins aggregate naturally. Maybe if your protein forms SDS-resistant aggregates this might happen (Irfan's answer; i.e. not denatured). There are harsher ways of denaturing protein that are harsher than SDS+boiling. I've heard very low concentrations of TFA+boiling is one of them.
Hi Pulu, have you added the protease inhibitor/s in extraction buffer, if not so then add the PMSF or cocktail inhibitor. During extraction try to maintain the 0-4 C temperature. i think your target protein is sensitive for degradation. load about 30-40 micrograms of freshly isolated protein on denaturing gel
Hi Pulu, have you added the protease inhibitor/s in extraction buffer, if not so then add the PMSF or cocktail inhibitor. During extraction try to maintain the 0-4 C temperature. i think your target protein is sensitive for degradation. load about 30-40 micrograms of freshly isolated protein on denaturing gel
I've made acrylamide gels a lot and I've had this issue on occasion. Use 15%, if possible, try making new buffers and building with completely fresh supplies (maybe the stock solutions are older (or if you share a lab maybe someone accidentally contaminated them). That, and the above suggestions should get you a clear result.
Also, make sure your power supply is functioning properly - I think a colleague had an issue just before a power supply failed where the amperage was fluctuating and not consistent.
I've made acrylamide gels a lot and I've had this issue on occasion. Use 15%, if possible, try making new buffers and building with completely fresh supplies (maybe the stock solutions are older (or if you share a lab maybe someone accidentally contaminated them). That, and the above suggestions should get you a clear result.
Also, make sure your power supply is functioning properly - I think a colleague had an issue just before a power supply failed where the amperage was fluctuating and not consistent.
Hi Pulu
On the contrary, I think ur denaturation is incomplete. Try to do 10 mins at 99 C.
Higher acrylamide would also help to distinguish small proteins.
Check your SDS buffer in which you make ur protein sample, it might need some extra DTT/b-mercaptoethanol. Often heppens with old buffers, then u add one two drops of them and its perfect.
Hi Pulu
On the contrary, I think ur denaturation is incomplete. Try to do 10 mins at 99 C.
Higher acrylamide would also help to distinguish small proteins.
Check your SDS buffer in which you make ur protein sample, it might need some extra DTT/b-mercaptoethanol. Often heppens with old buffers, then u add one two drops of them and its perfect.
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Some times smearing problem is assocaited with DNA in your sample, you can try shearing the sample with fine syringe it may help.
Some times smearing problem is assocaited with DNA in your sample, you can try shearing the sample with fine syringe it may help.
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Hey, I would try loading less of your protein sample first, as Dragana recommended.
Good luck!
Hey, I would try loading less of your protein sample first, as Dragana recommended.
Good luck!
More
VOTE
Hi! Pulu,
Run not less than a 13-14% gel for the protein your are trying to visualise (16kDa). Also, boil your sample for 9-10 mins before loading and load it immediately after preparing the sample without storing. Run your SDS-PAGE at a constant current of 20mA through out till the dye front reached the bottom of the gel.
Moreover, you can probe for your protein and see how it is visualised on western blot..
Good luck with your experiment
Hi! Pulu,
Run not less than a 13-14% gel for the protein your are trying to visualise (16kDa). Also, boil your sample for 9-10 mins before loading and load it immediately after preparing the sample without storing. Run your SDS-PAGE at a constant current of 20mA through out till the dye front reached the bottom of the gel.
Moreover, you can probe for your protein and see how it is visualised on western blot..
Good luck with your experiment
More
VOTE
Thank you very much for all the suggestions.
I have tried to increase the content of acrylamide from 10% to 15%, but it did not work. The smear still existed. At the end, I realized that the smear was caused by the function of the protein itself...
Thank you very much for all the suggestions.
I have tried to increase the content of acrylamide from 10% to 15%, but it did not work. The smear still existed. At the end, I realized that the smear was caused by the function of the protein itself...
More
VOTE
Hey, I agree with others saying you should use fresh buffers and higher percentage gel. Considering size of your protein, if I were you, I would probably use 20% gel (or at least 15%). Also, it is possible that you have high concentration of wanted protein and maybe you should try higher dilution of your samples (ie. 2x, 3x, not more) together with the one used in this picture and with that 15/20% gel.
One more, denaturation (if done with B-merc.ethanol) can be performed for 3-10 minutes on 95-100C, it does not make such a big difference. 7min 100C is very good (at leas in my experience), but I know that some people just leave their samples with b-merc.ethanol overnight at room temperature and denaturation also occurs (I habe not tried this). :)
Good luck
PS. Smear bands can also be caused by glycoproteins (the glyco-part).
Hey, I agree with others saying you should use fresh buffers and higher percentage gel. Considering size of your protein, if I were you, I would probably use 20% gel (or at least 15%). Also, it is possible that you have high concentration of wanted protein and maybe you should try higher dilution of your samples (ie. 2x, 3x, not more) together with the one used in this picture and with that 15/20% gel.
One more, denaturation (if done with B-merc.ethanol) can be performed for 3-10 minutes on 95-100C, it does not make such a big difference. 7min 100C is very good (at leas in my experience), but I know that some people just leave their samples with b-merc.ethanol overnight at room temperature and denaturation also occurs (I habe not tried this). :)
Good luck
PS. Smear bands can also be caused by glycoproteins (the glyco-part).
More
VOTE
To Heba Ebeed: Thank you very much for your answer! I did the protein extraction from sample for twice and they showed the same problem. Ok, so do you think the denaturation time is too long (I denaturated for 5 min at 95 C)? I will try your suggestion!
To Jan Piwowarski: Thank you very much for your answer! I will give it a try.
To Heba Ebeed: Thank you very much for your answer! I did the protein extraction from sample for twice and they showed the same problem. Ok, so do you think the denaturation time is too long (I denaturated for 5 min at 95 C)? I will try your suggestion!
To Jan Piwowarski: Thank you very much for your answer! I will give it a try.
More
VOTE
I have mainly seen this when my protein is associated with nucleic acids, just as Lokesh said. A quick check might be to determine the A260 nm:A280 nm ratio for nucleic acid contamination of your protein preparation. If nucleic acid is present then try shearing it (Lokesh's suggestion) and/or you could add DNase during lysis.
The other times I've seen odd upwards smears on protein gels when there is no nucleic acid involved is when the protein is very positively charged. They usually aren't so smeary. It could also be that your gel is melting during electrophoresis (similar to Shayne's answer). Was your buffer really hot at the end of your run and are other bands well resolved on your gel?
Note that some proteins are SDS resistant and other proteins aggregate naturally. Maybe if your protein forms SDS-resistant aggregates this might happen (Irfan's answer; i.e. not denatured). There are harsher ways of denaturing protein that are harsher than SDS+boiling. I've heard very low concentrations of TFA+boiling is one of them.
I have mainly seen this when my protein is associated with nucleic acids, just as Lokesh said. A quick check might be to determine the A260 nm:A280 nm ratio for nucleic acid contamination of your protein preparation. If nucleic acid is present then try shearing it (Lokesh's suggestion) and/or you could add DNase during lysis.
The other times I've seen odd upwards smears on protein gels when there is no nucleic acid involved is when the protein is very positively charged. They usually aren't so smeary. It could also be that your gel is melting during electrophoresis (similar to Shayne's answer). Was your buffer really hot at the end of your run and are other bands well resolved on your gel?
Note that some proteins are SDS resistant and other proteins aggregate naturally. Maybe if your protein forms SDS-resistant aggregates this might happen (Irfan's answer; i.e. not denatured). There are harsher ways of denaturing protein that are harsher than SDS+boiling. I've heard very low concentrations of TFA+boiling is one of them.
More
VOTE
Hi Pulu,
have you added the protease inhibitor/s in extraction buffer, if not so then add the PMSF or cocktail inhibitor. During extraction try to maintain the 0-4 C temperature. i think your target protein is sensitive for degradation. load about 30-40 micrograms of freshly isolated protein on denaturing gel
Hi Pulu,
have you added the protease inhibitor/s in extraction buffer, if not so then add the PMSF or cocktail inhibitor. During extraction try to maintain the 0-4 C temperature. i think your target protein is sensitive for degradation. load about 30-40 micrograms of freshly isolated protein on denaturing gel
More
VOTE
I've made acrylamide gels a lot and I've had this issue on occasion. Use 15%, if possible, try making new buffers and building with completely fresh supplies (maybe the stock solutions are older (or if you share a lab maybe someone accidentally contaminated them).
That, and the above suggestions should get you a clear result.
Also, make sure your power supply is functioning properly - I think a colleague had an issue just before a power supply failed where the amperage was fluctuating and not consistent.
I've made acrylamide gels a lot and I've had this issue on occasion. Use 15%, if possible, try making new buffers and building with completely fresh supplies (maybe the stock solutions are older (or if you share a lab maybe someone accidentally contaminated them).
That, and the above suggestions should get you a clear result.
Also, make sure your power supply is functioning properly - I think a colleague had an issue just before a power supply failed where the amperage was fluctuating and not consistent.
More
VOTE