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- เผยแพร่เมื่อ 27 พ.ค. 2024
- Okay now that you know how to cast an SDS-PAGE gel, how exactly do you use it? Here, Dan shows you how to set up your apparatus to run SDS-PAGE.
WAIT!!! Don't have a gel? Watch this video to learn how to make it: • How to Make an SDS-PAG...
Some important points to remember:
- short glass plate faces inwards
- remove the comb carefully
- load samples inside the wells, and do not over fill (usually 10uL of sample is loaded)
- fill the inner chamber to the TOP with buffer
- fill the BOTTOM of the outer chamber with buffer
- attach power supply: RED to RED and BLACK to BLACK
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When you dispense your sample and you push the button of the pipette, make sure you don't release the button of your pipette until the tip comes out, otherwise you will suck up the sample again. Just keep it pushed to the point where you don't expel air bubbles, and the sample doesn't get sucked back in. Hope that helps!
It's so satisfying when you load the samples in the wells! I would do a compilation video of this step
excellent videos!!! helps me out a lot when I'm in the lab... I'm more confident doing stuff in the lab after watching your videos! thanks
Excellent video. Thanks ... I haven't made an SDS-PAGE gel in over a year, so this was a helpful review.
I love how early 2000's this video feels
This is a very well explained video, it helped a lot! Thank you.
@Saran89 Sure, we'll put that onto the list of videos to work on. Thanks for the suggestion!
You place the samples directly in the wells as shown at 2:35 (but note that yes, the wells also contain some buffer because you filled the inner chamber to the top with running buffer)
thank u for making this video, it is very useful!!
Hi, exellent video. I have a question, how to tag a specific protein with antibodies?
Try to load your sample slowly - push out the sample slowly, a little at a time. That way you can avoid overloading. Also, don't fill up your lane to the top. Usually 10-20 microlitres is used in each lane, but that depends on the size of the wells.
What is the buffer used in inner and outer chamber?
Really helpful for us newbies ;) Thanks!
thank you so much for this video!
@dsjp2010 First question:
You don't need to calculate. Just take about 10uL of your protein sample and mix with 10uL of 2x loading dye. Boil for a few minutes. Give a quick spin in the centrifuge to bring sample to the bottom. Load either 10uL or all of the 20uL.
BUT it is recommended that you load about 1-10ug of protein when the sample contains 1 protein. If possible, just take different concentrations of your protein and load them to see which concentration gives you the best looking gel.
@dsjp2010 Second question:
No you don't have to have the same volume. Just be aware of how much protein you are loading, and that the more you load, the more intense your bands will be.
@zileburki There are several ways to deal with this problem:
1) You can use a plastic gel loading guide (you put it on top of the gel, and it shows the wells -- maybe your lab already has one)
2) You can draw the wells on the glass plate using a permanent marker (before assembling the apparatus)
3) You can also add some bromophenol blue dye to your stacking gel so that your wells are blue and easy to see (check your inbox for more details).
Otherwise, just practice! =)
wow! loved it! great work guys!:D
Hello, can you please make a video of protein quantification with BCA kit if you have not. It will be more logical first step before running the gel.
P.S your videos are great!! keep them coming....
Hello! First of all, thank you very much for this video! It was very helpful :)
I just have one question...when "topping off" the buffer in the inner chamber, is there a potential risk of the loaded samples "pluming" and running into adjacent wells?
Where do you place the protein samples, in the wells? or in the wells and buffer?
@labtricks How much bromophenol blue dye should I add to the stacking gel so that it becomes easier to see? Thanks
Turns out my tips were wrong, but I appreciate the feedback. I was wondering though how long the gels last after destaining. I stained with Coomassie blue and destained with water and we need to store them for at least two days. Do I need to treat with a fixing agent before storing them or will they be fine just in 4 degrees celsius?
Keep it in the destaining solution (water) and it should be fine. Your main concern is to make sure the gel doesn't dry out, so make sure it's submerged in the solution and keep a lid on the container. That should be good for a few days. To prevent anything from growing, you can change the water once a day, but it should be okay as is for two days.
@088zorba Load your sample slowly and watch your pipette tip so you can see when all your sample has come out. If you push out everything from your pipette too quickly, the air after your sample will also come out and you will get bubbles (you don't need to push the button all the way down....just push until your sample has come out). If you get bubbles, don't worry because they should float to the top.
You can add some bromophenol blue to your stacking gel buffer, and it will give the wells purple colour. Read the comments below as this topic has already been addressed. Hope it helps :) Good luck with loading!
Are the buffers in the inner and outer chamber the same pH? Thanks :)
Hi! I'm from Mexico and I have a trouble: When I try to take the sample with the pipette and loading in a lane I can't avoid that it push out strongly and this therefore contaminate the next lane, what can I do???
there is no need to put the whole apparatus in the ice ?
the best video in the world. thank you
When I take my tip out, it seems to suck the sample up too. Have you encountered that problem?
I tried this proceduce several times, but my sample got stock in the first gel, it can´t pass to the second gel, What i can do?
@vinnie1313 One trick is to wash the wells with distilled water after taking out the comb. (you can do this step over the sink, before assembling the apparatus). Another way to deal with this problem is to use a long gel loading pipette tip, and scrap out any acrylamide between wells before loading. Hope this helps =)
@labtricks but u didnt add buffer into the outer chambers?
Do u have videos of PAGE for DNA?
i do not use bio-rad apparatus i use something very old. once i have my gel ready and put it into the tank with the appropriate apparatus that it needs to attach to, and then fill the inner chamber with running buffer, there is leakage of the buffer into the outer chamber. how do i prevent this? what are the probable causes of the leaking buffer?
Hi cations and anions,
While loading my samples, the samples did not enter into the well but floated... Can you help me troubleshoot this?
Thanks
Thank you so much! very useful
What is the molecular mass of your protein? What % resolving gel are you using? (if your protein is very large, and you have a high % resolving gel, it is possible that your protein is getting stuck at the top of resolving gel)
@narcissus003 oh and in terms of buying it....i've never bought pre-cast gels, but i'm assuming you should be able to find 12.5%. you would have to check with the manufacturers about this question.
@ktochi297 Yes, in fact it is exactly the same buffer in both chambers.
@Niharika Gaur there is no need to put the apparatus on ice, as the SDS (should) denature any heat-sensitive folded molecules in the first place.
because he chose low voltage?
The biorad manual itself said the sds page system may have high temp if you use high voltage. The concern of high temp isn't for the sample, but for the sds-page system.
Hey nice video, whenever I remove my wells, I can see some acrylamide in them which blocks me loading any sample into them. what could be the reason and how to avoid it?
Fill a syringe with running buffer. Use the syringe with needle to push the remaining acrylamide out from well.
thnaks buddy
it was helpful
Thank you for stopping my tears 😩
what about the transfer proccess ? :)
Cool dude this is awesome
I have bad eyes and I can never see where the wells are. What can I do? :(
Excellent
What was the buffer used when you filled the inner and outer chamber?
Running buffer 1x, presumably
Probably TRIS-glycine, pH 8.3 (with 0.1% SDS) or something similar.
Hello, I hope you're still active on this channel... Would there be an issue if you use 10X running buffer instead of 1X? Also, I noticed you had a very thin pipette tip that seemed to allow you to insert the sample past the short window and access very close to the bottom of the well. I was using a regular tip and found I couldnt get past the short window so I had to gently release the sample from the point of the short window. It appeared that the dyed sample 'fell' down into the well ok but I'm wondering if the sample may have dispersed more than I could see and could have contaminated other wells? Thanks for the video!
you should use 1x buffer
from my personal experience, a regular tip (those for 2-20µLand 20-200µL) is enough to get into the gel well and that should be enough to prevent any contamination between the samples across the wells. Try to let the short glass plate facing you and tilt the pipette towards you a little bit. Though i am not sure about the plate thickness and the comb you are using. Being unable to touch the end of the gel well also prevents you from injecting the sample into the gel. XD
in the video they are using special gel tips which are thinner. commercially available
Thank you :)
@narcissus003 The recipe will vary based on what percent your stock acrylamide solution is. There is a really good online calculator that you can use to figure out your recipe based on what percent gel you want. Just Google "sds page calculator" and click on the first result (TH-cam won't let me paste the direct link here, sorry!)
Good luck and let us know if you have any more questions =)
How do we know what the desired percent is for a specific protein?
You are great
@Younusessa 0.003% of the dye in your stacking gel buffer should be enough. Check your inbox for more details.
@isfahanboy28 Go to 3:36 of the video (step 6) where Dan adds buffer to the outer chamber
prelab work... aint nobody got time for that
Hi everyone. Please anwser me, i should be running how much mA (current) with minigel in SDS PAGE?
30
Thank u
Can I join your Lab for internship?
nice!
what did the gel do to you for you to stab it like that in the top view? lol
Why do you need to fill the inner chamber, then load your sample and THEN fill the outer chamber ? Why can't you just fill the inner and outer chamber and then load your samples?
I did just like you. I guess it's all depends on how the handler likes it
it is helpfull
Legends are here from 2022__😂😂
180 V wasn't too high?
The video was too good, explaining clearly the entire process of electrophoresis...
What's the fun of sds page
Working on it all day and coming back to smears
Who else likes going $15k into debt for a youtube video ayyyyyyuyyyyyyyyyyy
What a bad memories brings me this technique....
It is a very nice video, but the focus is often on the background, that's very annoying.
Thank you! But please remove the music.