Hi Aleks,

since you are interested in a single protein of 60-70kDa size, why don't you simply use a 7,5% -10% polyacryamid gel?

You don't really need to see small proteins, right?

With this you shouldn't get in trouble running the samples and if you can't prepare the gels by yourself, these gel sould be cheaper than gradient gels.

If you are using brain homogenates, which kind of buffer do you use? The high lipid content might cause some problem during electrophoresis. One option would be to dillute your sample and check if the migration behavior gets better, while your protein-of -interest can still be detected.

If you do a wet transfer, try 100V for 1h, if you have a cooled transfer chamber or Ice-block. I usually use 100V for 1h for 1mm thick Gels and proteins of 50-100kDa. This sould also work and save time.

Best,

Kai

Hi Aleks,

first I have some questions to give you a helpful advice:

What size do your proteins-of-interest have?

Is the 4-20% gradient really necessary? Are you trying to separate proteins of similar size?

What kind of blotting technique do you use for blotting, semi-dry or tank-blot?

Usually it is the best to stick to manufacturers in the first place and I think it is normal, that the migration of your proteins "seem" to get stuck at a point were the gel pores get smaller and smaller. If you blot gradient gels you will have to optimes blotting time for your protein-of-interest and it is normal that the marker proteins from the low percentage part of the gradient are transfered faster than from the higher percentage part. Don't worry to much about your MW marker and check WB for protein of interest!

Best,

Kai

Hi Aleks,

I agreed with Kai, for both the questions (to understand better your problems) and advices (I cordially hate the precast gels!).

In my experiences I have problems with MW markers and gel precast, they often fainted so you can try to load double volume, for example.

You made a good choiche to follow the manufactures settings you are sure about the quality of your gel? I have problems with gel precast near experied date, or with different lot.

For you future experiment you need to understand if the problems are you gels, running settings, buffers (are hand made or not?), transfers settings or, your samples.

You do not need to waste you samples (especially if they are precious).

May you ask a sample to the coworkers that had good results? Can you use a positive controls?

The running settings are probably good and as Kai told you when the samples run in the gel you may have different speed across the gradient, and if the running buffer is not hot you do not need to cool it.

For the transfer settings I do not know which proteins you are study, if you need to see high and low molecular weight proteins sometimes it is difficult to choose a voltage and time settings correct enough for both the proteins. In this case try to focalized only on one protein and obtain a good results at a time.

I have more experience with tank-blot rather than semi-dry blot so told us your protein molecular weight (why you choose pre cast gradient gel) and which method you use.

Hi Aleks

besides the comments made before:

1- you only centrifuge your samples just one time (run @200V)? better to do it always--

2-your samples in which buffer?

from what you say for the first part (the run) you could either have genomic dna contamination or a running buffer not working well. Did you samples appear viscous? if yes, it is genomic dna. Anoher question, which running buffer are you using? why checking ph? usually if it is done ok no need.

so, first optimize the run, then we can talk bout the transfer.

In my experience, pre-cast gel (another brand) runs pretty well and without any fuss...

Try doing a ponceau stain at the end of the western transfer process, before you put the blot in the blocking solution. This will tell you if the protein has been transferred to your membrane or not.

Is anyone else at your institution running this gel system? I understand your frustration, it can be depressing. I would focus on getting the gel to run properly first before you worry about blotting. What is in your running buffer? When the proteins appear to stop running, what happens to the voltage and current? Don't worry about samples, use coloured markers. You can get it to run. I have used precast gels for 15 years and would never go back to making my own. It will work.

I would definitely go about this very systematically.

1) I would run maybe just stds in a couple of lanes.

2) I would make sure there is no leak in the running system and the buffer levels are optimal during the entire run. You may have to monitor this very closely during the entire 45 minute period.

3) If anyone else has had success with this and they have a buffer use this running buffer as opposed to yours and see if this helps.

This will ensure the problem with the running setup. Once this is resolved;

1) Setup your transfer ( I personally have always had a lot of success with a wet transfer rather than a semi-dry) and make sure the transfer buffer has the right amount of methanol.

2) You can increase the time by a little for transfer and since your markers are prelabeled you will visualize them easily.

3) I will also make sure that the transfer stack is compact and will have efficient current passing through.

Hope this helps.

Hi, Thank you so much everybody for your responses

I am doing a wet transfer

It is just me doing the transfers in the lab, The other colleague who is doing this is at another institution. We use the same recipe for running and transfer buffers.

I make buffers 1x every time fresh.

(Running: 1L 14g glycine, 3g tris, 1% SDS pH 8.3; Transfer: 2L 28g glycine, 6g tris, 20% methanol pH 8.4)

I ve heard mixed reviews about adjusting the pH, mostly that it is not necessary, but my advisor told me I should do it anyhow

There are no leaks in the system for both the running step and the transfer step

I use two lanes to run the markers. Both in the last lanes

The proteins and the samples dont per se stop running, they just significantly slow down in the middle, the voltage and current stay the same. I do agree that it is probably to the gradient of the gel matrix, Ive just never work with this type before and wasnt sure if this is normal.

On my last run at 200V, the voltage reached 200 at about 15 minutes after I started and then remained constant. Not too sure about the current, I think it fluctuated a little but not by much. For transfer, the voltage stay constant the entire time (most of the runs, sometimes it does fluctuate a little)

I dont think it is the integrity of my samples that is the question. I think it is either the running buffer or the voltage I am running at. The manufacture is asking to run for 200V with Tris Hepes buffer since it is the Tris Hepes gels. I use Tris Glycine buffer...

So this gel is for 205-2.5kDa. My protein is about 60-70kDa.

According to the migration chart for this gel, my samples will stop before half way which would be the migration point for 45kDa.

So what was probably happening is that my samples stalled because they were too big for the pore size. Since my Voltage is so high they were pushed through and hence caused all the funky band distortion??!! I stop at about 3/5 of the gel

If so, I guess what I dont understand is that how come the dye front would just not travel all the way. I working with mouse brain homogenates and cell culture samples. Wouldnt they have some proteins of smaller size that would run to the end of the gel? Is that a silly question

My MW standard is 180-17kDa. Sometimes the lower MW bands are not sharp and dont separate well. Possibly due to the high current. I think I had a better luck in separation under 100V. Although not always

How can I make sure that during transfer all of my protein bands transfer?

I transfer for about 2-2.5 hours and sometimes I still see the bands of MW marker on the gel. I image my blot with ECF and I get plenty of signal, although I wonder if I leave some of the protein on the gel, some of the MW bands faintly reside on the gel? I guess I can do gel staining.

If I do increase the transfer time, how do I make sure I dont over transfer and how would I know if I over transfered. (PonceauS for the blot and Comassie for the gel)

Hi Aleks,

since you are interested in a single protein of 60-70kDa size, why don't you simply use a 7,5% -10% polyacryamid gel?

You don't really need to see small proteins, right?

With this you shouldn't get in trouble running the samples and if you can't prepare the gels by yourself, these gel sould be cheaper than gradient gels.

If you are using brain homogenates, which kind of buffer do you use? The high lipid content might cause some problem during electrophoresis. One option would be to dillute your sample and check if the migration behavior gets better, while your protein-of -interest can still be detected.

If you do a wet transfer, try 100V for 1h, if you have a cooled transfer chamber or Ice-block. I usually use 100V for 1h for 1mm thick Gels and proteins of 50-100kDa. This sould also work and save time.

Best,

Kai

Hi Aleks, i don't use Tris Hepes but always Tris Glycine with hand made and precast gels, I do not have experiences but if the manufactures ask for a particular buffers I would try to make Tris Hepes to run my precast gel!

I think that you have to resolve the running problem before the transfer one.

A suggestion:

If your protein is 60-70 KDa and you can find a protein to normalized between 40-50 KDa (with your samples it is not a problem), I think that you have to make an hand made gel (10% for example), with your Tris Glycine buffer (I don't agree with the pH, issue but all the "advisors" have their way to work!) and run your sample without problems. Ask your advisor if you can make this experiment in my opinion you will probably have good results.

Aleks, I've checked on the website of the NuSep vedor...so..two important things:

1-the gel does not contain SDS so it came from the buffer...tris-gly buffer has a bigger charge carrier (gly) than hepes buffer and it brings together the sds..so it is probably the cause you see the stopping of the gel, also demonstrated by the fact that it takes time to reach 200V...

2-also your recipe for tris-gly is slightly incorrect: 14,4 g Gly, 2,9 tris and 1%sds. and you do not pH this solution because:1-sds screws the pH electrode giving weird readings 2- if your pH is not correct, then the solution is not ok and if you change the pH you change the ionic strength and so the run changes....

hope this helps, first optimize the run and then we can talk about the transfer! :) good luck!

First try to run a good gel...if you are not confident about how your gel run, then simply before going for a transfer, first stain your gel after running with coomassie.

Second, if you are running a mini gel then u can run at 25mAmp for 2 hour (depends when your tracking dye start coming out).

Third, just add 1% of 10% SDS in your transfer buffer and let your transfer run at low voltage (say 25Volts) for overnight (you can also put for 14-16 hours). But if your protein are less then 20kDa then keep for overnight otherwise it can go out (simply the funda of timing is volts X timing=256, for example 16 volts for 16 hours=256 ).

Also, activate your PVDF membrane in some methanol on slow rocker for 2-3 minutes, after that keep it in transfer buffer to equilibrate.

Fourth, after transfer stain your membrane in ponceau or fast green.

I thnk it will works!...keep writing...Best