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SDS-PAGE Gel Electrophoresis Forum SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


SDS-PAGE Problems: I'm getting desperate!

SDS-PAGE Problems: I'm getting desperate! - SDS-PAGE Gel Electrophoresis Forum

SDS-PAGE Problems: I'm getting desperate! - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


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  #1  
Old 11-30-2010, 09:43 PM
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Default SDS-PAGE Problems: I'm getting desperate!



I have been trying to run SDS-PAGE on my samples for over a month now.

I cannot post an image, but I will describe it: Every protein above about 50 kDa is gone. Nothing there at all. There is a front on the lane that looks like
a camel's hump. Not counting that, the way it goes from normal bands to absolutely nothing is very abrupt. The ladder has been of varying quality, going from looking fairly normal to being blotched and warped. More recently, the blotched/warped version seems to be the prevailing result. I have a BSA sample that I ran on the gel, but it vanished along with the other heavier proteins.

Here is what I did:

The gel is 10% and was made from scratch by myself. (The u = greek letter mu, as in "microliters")

My recipe for one 10% gel. Everything was freshly made today.
Resolving Gel
30% Acrylamide (29:1 Bis) - 1.34 mL
double distilled H20 - 1.14 mL
8.8 pH 2M Tris + HCl - 1.45 mL
10% SDS - 40 mL
10% APS - 35 uL
TEMED - 3.5 uL
Stacking Gel
30% Acrylamide (29:1 Bis) - 200 uL
double distilled water - 1.098 mL
6.8 pH 2M Tris + HCl - 188 uL
10% SDS - 15 uL
10% APS - 30 uL
TEMED - 3.0 uL

I clean the glass and ceramic plates well every time I use them, wiping them down with detergent, rinsing with distilled water and squirting on some ethanol to help dry. I make sure the plates are dry before casting. I mix up the resolving gel as above, making sure that the two Tris solutions are NOT mixed up and stirring well between each ingredient addition. After the ingredients are mixed, I will pour them into the caster with a p1000 pipet, making sure the resolving gel ends 1 cm beneath the bottom of the comb. I will put on about 1 mL of water-saturated butanol and wait for the gel to polymerize (15-20 minutes, usually.) After the gel is done, I will pour off the butanol, rinse with water, then leave to let the water pour out, blotting up any excess water if necessary. The stacking gel is made as per the above recipe and is added to the top of the resolving gel, then the comb is added and left to polymerize. Afterward, I add my standard and samples.

My samples are whole cell lysates from E. coli. I take 1 OD worth of cells, spin them down, pour off the media and add 250uL of 1x SDS-PAGE loading dye (the kind with the bromophenol blue, glycerol, SDS and water. I don't have the recipe here, but I don't think it's the problem anyway, since older samples that I did not make look similar to the picture above.) I am sure to boil the samples for at least 5 minutes, vortexing and centrifuging down before loading. I load 10 uL of each sample.

I use 1x Tris, glycine, and SDS running buffer. (I do not think this is the problem, as I have used 3 different buffers with no difference in performance.) I run it with 100V until the dye-line hits the resolving gel, when I turn up the voltage to 150V. It runs on the stacking gel usually for about 30 minutes and on the resolving gel for about an hour.

I have tried using someone else's reagents. I have tried remaking my own reagents. Neither seemed to help. I am very careful when making the gels, being sure not to spill or induce contamination to it. I make sure everything is clean before I use it.

I am seriously running out of ideas, as well as sanity. Any help you can give me would be appreciated. Thank you!
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Old 12-01-2010, 12:53 AM
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Default Re: SDS-PAGE Problems: I'm getting desperate!

Update: I ran some samples on a pre-cast, premade gel and got the same results. This narrows it down to my samples or my buffer. I don't think it's the samples, however, because I've recently retried old samples I knew worked and they turned out just as crummy. A sample of pure BSA didn't even show up. This eliminates it to the buffer, but despite the fact that I used someone else's AND remade my own, it still didn't work. I'm at a loss, really.
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Old 12-01-2010, 06:58 AM
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Default Re: SDS-PAGE Problems: I'm getting desperate!

Hola, if the phenomenom occurs with commercial pre-cast gels, you have think that the problem is with the samples or the running buffer,but comparing the recipes with mines there are differences( I suppose that the amount of SDS in the resolving gel is 40ul).Check in internet or in any manual or ask people without your problems by the recipes, and compare them. If you want I could answer again with them.
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Old 12-01-2010, 06:00 PM
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Default Re: SDS-PAGE Problems: I'm getting desperate!

Here, I added this image, if that helps anything. This is what the majority of my gels look like.
Attached Images
File Type: jpg 11-30-10 SDS-PAGE troubleshoot.jpg (84.3 KB, 34 views)
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Old 12-03-2010, 02:44 PM
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Default Re: SDS-PAGE Problems: I'm getting desperate!

Well, here I am for another update.

Firstly, I ran a gel using a precast gel and an apparatus and buffer that was not my own and the results came out beautiful. This proves that the problem is most likely not in my samples, so that can be eliminated once and for all.

The image is attached as "12-1-10 Precast Gel SDSPAGE.jpg"

Here is another gel I ran at the same time, with my own materials and someone else's buffer - named "12-1-10 Selfcast Gel SDSPAGE.jpg"

Then I ran one more gel, this time with my own reagents and the buffer from the same person who offered me the precast gel and their own buffer. Named "12-2-10 SDS-PAGE troubleshoot.jpg"

Although it wasn't perfect, I thought that maybe whatever problem it was was overcome. My PI suggested that it was probably the fact that the buffers were leaking, so I sealed the gel plate to the running apparatus with some petroleum jelly. I also used the running buffer I made myself earlier this week. I got back to work on the proteins I had purified and ran a gel for two of them. The name is "12-2-10 Tau Purification 3.jpg" There is no image for the other protein because that gel, despite being cast from the same aliquot of solution, being run on the same apparatus and with the same buffer, it came out COMPLETELY BLANK. That is, other than a few smudges near the bottom of the gel.

The thing that's bothering me the most here is the wild and nonsensical inconsistency. None of this seems to make any sense. There are lanes that look fairly good and lanes that look like total junk on the same exact gel! I know it can't be the samples. I would say that maybe it's the water (actually writing this while waiting for a new bottle to autoclave) but it doesn't explain the somewhat normal gel + COMPLETELY BLANK gel combo I got yesterday.

I'm really out of ideas. This whole ordeal is really causing me a lot of unnecessary stress. I am way behind as far as gathering data goes and someone who has far less experience than me is getting much more data with a few half-days a week than I am with 5 whole days! Please, if anyone can give me even a hint I would be very grateful!
Attached Images
File Type: jpg 12-2-10 Tau Purification 3.jpg (89.8 KB, 13 views)
File Type: jpg 12-1-10 Precast Gel SDSPAGE.jpg (77.8 KB, 19 views)
File Type: jpg 12-1-10 Selfcast Gel SDSPAGE.jpg (61.8 KB, 16 views)
File Type: jpg 12-2-10 SDS-PAGE troubleshoot.jpg (82.3 KB, 20 views)
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Old 12-03-2010, 10:09 PM
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Default Re: SDS-PAGE Problems: I'm getting desperate!

Well, I think I finally figured it out!

Ran it on a different apparatus with everything else different and it worked beautifully!

Still, I find it odd how my lab mate can use one of the same apparatuses and get great results. It doesn't make a whole lot of sense... I may fiddle with that later. :/
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Old 12-07-2010, 10:57 PM
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Default Re: SDS-PAGE Problems: I'm getting desperate!

So I don't think it's the apparatus, because I've used the new one and while generally the results are better, there have been several gels that haven't come out so good, including one that looks just like the old ones. Anybody else have any ideas? There's been over a hundred views on this thread and nobody has a clue?
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  #8  
Old 12-08-2010, 09:45 PM
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Default Re: SDS-PAGE Problems: I'm getting desperate!

Your recipe for the SDS-PAGE gels looks vastly different from one that I use. Doing the calculations, you are using roughly double the amount of Tris in the gels than what my recipe calls for. If you'd like to try a different recipe, here is mine for a 10% gel. I believe this is a fairly standard recipe for gels from what I have seen:
Separating (makes 10mL):
H2O = 4mL
1.5M Tris-HCl, PH 8.8 = 2.5mL (note the different M of the Tris buffer I'm using-this is a 4X stock)
30% Acrylamide = 3.3 mL
10% SDS = 100 uL (final concentration 0.1%)
10% APS = 100 uL
TEMED = 5 uL

Stacking (makes 10mL):
H20 = 6mL
0.5M Tris-HCl ph 6.8 = 2.5 mL
30% Acrylamide = 1.3 mL
10% SDS = 100 uL
10% APS = 50 uL
TEMED = 10 uL

Running buffer is (final concentration of each component):
25 mM Tris-base
190 mM Glycine
0.1% SDS

I have found with precast gels that you have to watch what they are made of and what buffer to use with them. For example, a Tris-glycine based running buffer can give weird results if used with a Bis-Tris gel (should use a MOPS or MES based buffer instead).
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Old 12-08-2010, 10:09 PM
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Default Re: SDS-PAGE Problems: I'm getting desperate!

Alright, so I think I've finally figured it out- my stacking gel was not set enough! I think that when I started the gel running, it would continue to set, messing things up. I'll probably confirm this tomorrow.
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Old 01-07-2011, 03:00 PM
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Default Re: SDS-PAGE Problems: I'm getting desperate!

Hello!

This happened to me when I ran my gel for the first time and yes I could figure out the reason and its alright with me now.The problem was with the gel it was not 10% so i had the problem.

I hope the problem is with the resolving gel that you use.
this is what i use for 10% resolving gel of 10ml
H2O- 4ml
30% acrylamide-3.3ml
1.5M Tris ph 8.8-2.5ml
10%sds-0.1ml
10% APS- 0.1ml
TEMED-0.004ml

Stacking gel for 10ml is

H2O- 6.8ml
30% acrylamide-1.7ml
1.5M Tris ph 6.8-1.25ml
10%sds-0.1ml
10% APS- 0.1ml
TEMED-0.01ml


try out with this
hope it works for you

-shilpa.
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