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SDS-Page issue


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#1 anatg

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Posted 09 November 2009 - 01:16 AM

Hello

I am targeting HSP70 and since I was having some issues with the blotting, I decided to Coomasie Brillian stain the gel after the blot to check its efficiency. I loaded the gel like this:
1.Marker
2.HSP70 pure standard
3.sample 1(liver homogenate) total protein
4.sample 1(duplicate)
5.sample 2(liver homogenate) total protein
6. sample 2 (duplicate)
7. negative control (only sample buffer)
(etc as mirror until the 14th lane since the gel was to cut in 2 to test different antibodies)
I realized that my blot wasn't efficient since the proteins were still in the gel, but what I found strange was that the marker and positive control were in the gel, but from the liver homogenate ONLY the 70kD (which I assume is actually my target protein) were in the the gel. I haven't stained the membrane to see if something passed or not (although at least half of the marker passed). My question is, in a case of a bad blotting when the proteins stay in the SDS gel, a total protein homogenate should have many lanes and not only and exclusively on the 70kD. I use as extraction buffer 1 ml (of 1mMEDTA 1mM PMSF in PBS ice cold with protease inhibitor cocktail) for 100 mg liver (of fish by the way), homogeneized, centrifuged, and supernatant stored at -80C until sds page/western blot.

Any thoughts about this are very welcome since I think I am missing something very basic here...

My best regards to all

Anatg

#2 DrRob

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Posted 09 November 2009 - 03:38 AM

Hello

I am targeting HSP70 and since I was having some issues with the blotting, I decided to Coomasie Brillian stain the gel after the blot to check its efficiency. I loaded the gel like this:
1.Marker
2.HSP70 pure standard
3.sample 1(liver homogenate) total protein
4.sample 1(duplicate)
5.sample 2(liver homogenate) total protein
6. sample 2 (duplicate)
7. negative control (only sample buffer)
(etc as mirror until the 14th lane since the gel was to cut in 2 to test different antibodies)
I realized that my blot wasn't efficient since the proteins were still in the gel, but what I found strange was that the marker and positive control were in the gel, but from the liver homogenate ONLY the 70kD (which I assume is actually my target protein) were in the the gel. I haven't stained the membrane to see if something passed or not (although at least half of the marker passed). My question is, in a case of a bad blotting when the proteins stay in the SDS gel, a total protein homogenate should have many lanes and not only and exclusively on the 70kD. I use as extraction buffer 1 ml (of 1mMEDTA 1mM PMSF in PBS ice cold with protease inhibitor cocktail) for 100 mg liver (of fish by the way), homogeneized, centrifuged, and supernatant stored at -80C until sds page/western blot.

Any thoughts about this are very welcome since I think I am missing something very basic here...

My best regards to all

Anatg


Hiya, not sure what exact protein you're working with but is it possible that the protein is highly charged in relation to the others? A strong positive charge (imparted by many lysines for example) could theoretically alter the mobility of the protein during transfer. Also, it is possible that some of your standard is "leaking" into the adjacent lane...

#3 bob1

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Posted 09 November 2009 - 05:24 PM

That is the weakest lysis buffer ever, I suggest adding some SDS or other detergent (e.g. triton X-100, NP-40, nonidet-p40 (not the same thing), deoxycholate) as you probably haven't solubilised much of the proteins in the samples.

#4 anatg

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Posted 09 November 2009 - 07:58 PM

Thank you very much for your answers

The protein I am aiming is the Heat Shock Protein 70, and the buffer I am using to extract is indeed a very week buffer. Nevertheless, this is the used buffer referred by other authors for the same conditions of my experiments, and it is not the first time i am using. I know the buffer works because I already did the very same protocol and I got very good valid results. The problems started when my laboratory bought a new and different power machine for the blotting device. I have serious trouble to manage the voltage/amperage/time relationship without having errors. I just couldn't get any bands on membrane from that moment on...can only see the marker. And now I am doing a complete troubleshooting, and when it came the time to test ow many proteins are still in the gel (since it seems the blotting was not well performed) I can only see the ones the 70kD area. It would be explainable if i.e. 50% of the total proteins are the 70kD ones that are overexpressed, and all passed but only half of the amount of 70kD passed....but, in that case, the positive control (which is low concentration pure protein would had passed to the membrane, right?) should not be in the gel anymore...I think....
Anyway, i am running it again, continuing troubleshooting and I will see what will happen...i will also increase the time of blotting.

Thanks anyway

My best regards

Anatg

#5 mdfenko

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Posted 10 November 2009 - 07:27 AM

if all you are seeing left in the gel is the ~70kDa proteins then longer transfer will not improve the situation. it may even make it worse.

you are probably blowing your proteins through the membrane. the output of your new power supply appears to be greater than that of your old supply (either the new one is putting out more than indicated or the old one was putting out less than indicated).

you can test for blow through by putting a second membrane behind the first. you can also try a smaller pore membrane.

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#6 Feelcontraire

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Posted 18 November 2009 - 05:22 PM

Hello

I am targeting HSP70 and since I was having some issues with the blotting, I decided to Coomasie Brillian stain the gel after the blot to check its efficiency. I loaded the gel like this:
1.Marker
2.HSP70 pure standard
3.sample 1(liver homogenate) total protein
4.sample 1(duplicate)
5.sample 2(liver homogenate) total protein
6. sample 2 (duplicate)
7. negative control (only sample buffer)
(etc as mirror until the 14th lane since the gel was to cut in 2 to test different antibodies)
I realized that my blot wasn't efficient since the proteins were still in the gel, but what I found strange was that the marker and positive control were in the gel, but from the liver homogenate ONLY the 70kD (which I assume is actually my target protein) were in the the gel. I haven't stained the membrane to see if something passed or not (although at least half of the marker passed). My question is, in a case of a bad blotting when the proteins stay in the SDS gel, a total protein homogenate should have many lanes and not only and exclusively on the 70kD. I use as extraction buffer 1 ml (of 1mMEDTA 1mM PMSF in PBS ice cold with protease inhibitor cocktail) for 100 mg liver (of fish by the way), homogeneized, centrifuged, and supernatant stored at -80C until sds page/western blot.

Any thoughts about this are very welcome since I think I am missing something very basic here...

My best regards to all

Anatg



True, that buffer is very mild, if you just pretend to perform sds-PAGE get used to lyse your samples in loading buffer+inhibitors.

To improve your transfer you may take a look at his: http://www.protocol-...showtopic=10851 .

Is heat shock protein heat labile? If so instead of using heat for the transfer use DHEBA for polymerizing the gel.




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