How to quantify gel bands in imageJ | common quantification mistake

[@阮丛霖]

Thank you for your video. Your sample picture is clean. What should I do to deal with dirty background? Some background area is even stronger than the band.

[@AliceritaE]

Draw an ROI on thr background and measure it. Then go to PROCESS/Math/Subtract (enter the value from the ROI that you measure) then click okay. This step should remove the background

[@olalukmanganiyu3425]

This is the most helpful video of all i have watched today. Thanks a lot!

[@AliceritaE]

Glad you liked it, thanks for watching

[@t0rchbearer]

Thank you so much! This video is very helpful. I used it to quantify my gel electrophoresis bands from CRISPR deletions.

[@t0rchbearer]

also you've earned my subscription! Keep making more content like this!

[@AliceritaE]

You're welcome, I'm delighted you found it useful.

[@AliceritaE]

Thanks for subscribing

[@abayomiadeleke7765]

This accent is so soothing. Thank you! 😀

[@AliceritaE]

Glad you think so!

[@job506]

The accent is simply so amazing; she's my sister.

[@AliceritaE]

Hi JOB

[@job506]

@@AliceritaE Hi my sister.

[@evelynjunaviles2582]

hi thanks for the video Im trying to learn how to measure my electrophoresis. So, while doing this, you don't have to remove the background? it isn't necessary? I just go straight to the band and measure ?

[@AliceritaE]

Hi Evelyn, Yes, you don't need to remove the background.

[@evelynjunaviles2582]

@@AliceritaE thank you. im excited. lets see how it goes !!

[@AliceritaE]

@evelynjunaviles2582 😊

[@bismagulzar9959]

Thank you so much for this vedio Love from Kashmir #

[@AliceritaE]

You're welcome Bisma, greetings from England

[@adrianaramirez8186]

Thank you so much, I have a trouble, when I click select next lane it duplicates and goes back to the 1st selected lane, so I have 2 selected lanes but it´s the same, how could I solve it? thank youuu

[@AliceritaE]

Hi, when you duplicate it and move it to the first lane, drag it to the second lane and continue like that.

[@kishorkumarkeekan8649]

Hi ..how to select the same rectangular box (with the same dimension, which is important) again for the next lane, that can be moved to rest if the lanes ....thank you

[@AliceritaE]

If you follow the step-by-step I showed in this video, the rectangle should be automatically duplicated for you to move to the next lane.

[@kishorkumarkeekan8649]

@@AliceritaE yeah.....I saw some of your previous reply to comments. 👍It was Ctl + D. Your video was very useful. Thank you ..regards. ☺️

[@AliceritaE]

Awesome! Thank you for reviewing other comments for answer. Cheers!

[@judith4294]

thanks for the helpful video. how report the intensity in what units? and do you know how to perform the analysis if i perform a qualitative rt pcr, where i have my bands for the control gene and the interest gene in samples control and treatment? thanks in advance.

[@girlcode3564]

Your video was very helpful, thank you.

[@AliceritaE]

You're welcome 😊

[@oghenefegorimieye4380]

Thank you so much for this video! Please how do I duplicate the rectangle. When I try it just starts a new rectangle

[@AliceritaE]

Hello, you can click on crtl + D to duplicate the rectangle.

[@oghenefegorimieye4380]

@@AliceritaE thank you! I tried that, it works for the first 2 lanes (,2,3) but after that it makes the lane I marked as 3 and tried to duplicate become a black box and then 3 moves over

[@AliceritaE]

That's strange! The bands have to be in the same lane. If you change the lane, that may happen. I will troubleshoot tomorrow and record a new video if the process has changed.

[@wukki99]

Thank you this is very helpful for me who is the first to use. :>

[@AliceritaE]

You're welcome.

[@2women88]

very useful video, thank you very much, do you know how to do the further excel calculations and graph, please?

[@AliceritaE]

First normalise your data by using by using smallest value to divide all the samples. To get the first ratio. To get the second ratio, divide the protein of interest by the reference sample (use the value of ratio one)

[@AliceritaE]

There is a tutorial online, here is the link kzbin.info/www/bejne/b5fMoY2lr76opdU

[@2women88]

@@AliceritaE I really appreciate your help and reply, but my supervisor want the mean and mean reciprocal method for quantification which I could not find until now !

[@AliceritaE]

Did you check out yhe link i sent to you. the lady calculated the mean.

[@2women88]

@@AliceritaE yeah, I checked it and it is different from what I should do 😓

[@yuxinsong-h2q]

hello，thank you for your video.I am wondering when I select the second lane,it automatically goes back to the fist line position.How could I do,I can choose next lane?Thank you

[@AliceritaE]

Hi Yuxin, After you have selected the first lane and marked it as first, when you move it to the second lane, you need to mark it as next until you get to the last one.

[@ritasm5843]

thanks for the tutorial🙏

[@AliceritaE]

You're welcome 😊

[@lamiachowdhury4636]

Hello! May I know what format the image of the gel should be in? Does Jpeg work? Also, does the same process apply to gel electrophoresis results?

[@AliceritaE]

Hi Lamia, the Gel image can be in any formats

[@alikivasilakou6145]

Hello! I was wondering the same, could we use this method for gel electrophoresis results?

[@AliceritaE]

Hi Aliki, yes you can use this method. The image I used in the tutorial is jpeg, it can also be tiff, it doesn't matter.

[@alikivasilakou6145]

@@AliceritaE thank you!!

[@AliceritaE]

You're welcome

[@yulywu4359]

Hi. Thanks for the great video! May I bring up a question? Is the area measuring the size of the bands or their signal intensity? Thanks!

[@AliceritaE]

Hello! The measurement is for the intensity

[@yulywu4359]

@@AliceritaE Thank you so much for the clarifications!

[@AliceritaE]

You're welcome 😊

[@ebunabeke6094]

informative as usual

[@AliceritaE]

Thanks

[@hiranasir2069]

Mam how do you move these plots downward so you can draw a separate line in each peak? please tell me

[@AliceritaE]

You can press the down arrow key in your keyboard to move to the next peak.

[@hiranasir2069]

@@AliceritaE yes mam am trying but its not working i don't know why

[@AliceritaE]

I'll check tomorrow morning

[@hiranasir2069]

Mam my image is an RGB color. Is it ok or should I convert it into a grayscale image?

[@AliceritaE]

Please convert it to 8bit image first

[@isabellephieler3329]

very helpful! thank u for the tutorial! :)

[@AliceritaE]

You're welcome!

[@makchitgaladima6620]

Hi, I have a western blot image that is so faint on an x-ray film. After plotting the bands, the resulting images were kind of zigzag with no specific peak but several. How do I get to the area?

[@AliceritaE]

Hello Makchit, You can try to enhance the gel image. Go to imageJ PROCESS// ENHANCE CONTRAST then try plotting the area again

[@makchitgaladima6620]

@@AliceritaE Okay, I will get back to you, thank you.

[@AliceritaE]

You're welcome 😊

[@brookestem1513]

What do I do once I have the area to find the protein concentration?

[@AliceritaE]

If you follow the video, you will get the value of the protein concentration. You need to do some calculations using the blade of the control to work out the ratio of the protein.

[@manikrpc]

Thanks! Well explained!

[@AliceritaE]

Glad it was helpful! thanks for watching

[@LydiaIglesias-q6i]

Fantastic video, thanks!! I am trying to do the same but when I select the second lane (going horizontally), it automatically goes back to a vertical position. How can I force it to go horizontally?

[@AliceritaE]

Hi Lydia, by default, the peak measurement data for gel quantification is given as a long vertical graphs.

[@sagirmustapha1828]

Thank you so much for the video, please how can we use excel to calculate and plot using graph pad prism, please???

[@AliceritaE]

I will prepare a tutorial at the weekend to address this. mote people have asked this question

[@AliceritaE]

Hi @SAGIR I founf a tutorial that explains the calculation very well. Here is a link to watch it. kzbin.info/www/bejne/a6TZaGh7n86omZY

[@kinkpelionel3287]

thanks Alice I would love your help in my research I don't know how to contact you. Your video has enlightened me a lot on certain points.

[@AliceritaE]

You're so welcome! I am glad you found it useful. you can contact me via e-mail of linkedln from my youtube profile

[@kinkpelionel3287]

@@AliceritaEok thank you for your reply. Blessing. May I have your email, please?

[@AliceritaE]

[e-mail]

[@j0hn0b]

thank you so much!

[@AliceritaE]

You're welcome!

[@joyzhou8179]

Thank you very much for the video! It help me a lot.

[@AliceritaE]

Hi Joy, I'm glad you found it useful. thanks for your kind feedback

[@dianaa.valencia3910]

Does it matter the image type? I mean 8, 16, 32 bit ??

[@AliceritaE]

no, it doesn't matter. Converting to 8 bit helps to convert shaded or coloured gel into black and white image.

[@Jessica-xw1rb]

Hello, can i use this method to estimate the mass of the band and does this method take into account of background readings?

[@AliceritaE]

this method unifies the background readings for all the samples (provided the same paramerters was used). By mass, do you mean the molecular weight? I found a guide eon Bio-RAD www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6210.pdf

[@Jessica-xw1rb]

Alicerita Thank you so much 😊

[@AliceritaE]

You're welcome 😊

[@LaboratoriumFarmasiTerpadu]

is it possible to rotate the lane to see pick of my gel?

[@AliceritaE]

It might be possible. Give it a try

[@makchitgaladima6620]

Hi, my question is how do I quantify proteins on X-ray films

[@AliceritaE]

HellO MAKCHIT, You need to scan the xray film to a digital photo format. And then follow the steps I used in the tutorial to quantify your protein.

[@makchitgaladima6620]

@@AliceritaE Yes, that has been done and followed the tutorial. I plotted the lanes but the peaks were not giving me appreciable values, so I tried densitometry where I calculated the ratio of the protein on x-ray film: the value of total protein on each lane on SDS gel stained with Coomassie. Do you think I have done the right thing?

[@AliceritaE]

the ratio measurement is corrrect. did you include a control in your analysis. that gives extra detail regarding about the sample

[@jasonwang9528]

Thanks for your video! It is really helpful!

[@AliceritaE]

You're welcome, glad you found it useful

[@firebolt8907]

Informative video... 👍 #AVMCreations

[@AliceritaE]

Thanks

[@solarium93]

Thank you, for this tutorial This is very helpful!!

[@AliceritaE]

You're welcome!

[@cc-vd3oq]

fantastic video! Thank you for making it!

[@AliceritaE]

It's a pleasure. Thanks for your kind comment

[@studentIndia-on3en]

You are awesome.thanks

[@AliceritaE]

You're welcome 😊

[@Shaylen721]

How do you duplicate on the keyboard when you make the first square? Because they have to be the same size

[@AliceritaE]

Press T

[@AliceritaE]

It added the location to a ROI manager from which you can then click on it to paste it on the new images. Keyboard shortcut T to save ROI to a manager Shift + Control + E to paste

[@aniebietessienpgs6903]

what version of image j did you use here?

[@AliceritaE]

Hi Aniebiet, My versionis currently v1.53i. There is a new update for v1.53k, which I will install later

[@hudsonalakonya6808]

Thank you so much.

[@AliceritaE]

It's a pleasure 🙏 Thanks for watching 🙂

[@lucpas157]

lines disappear, any solutions?

[@AliceritaE]

You mean the line from thr plot disappeared. Try and repeat the step

[@AliceritaE]

Any luck?

[@lucpas157]

@@AliceritaE Problem solved, thank you

[@AliceritaE]

Excellent

[@hafiezlukmandwiputro3599]

How to duplicated the rectangle?

[@AliceritaE]

kzbin.info/www/bejne/gZu8oKWladyDg5Y

[@francescosilvestro2092]

Thanks a lot.

[@AliceritaE]

You are welcome Francesco

[@kinkpelionel3287]

Thank you a lot

[@AliceritaE]

yes you can quanitfy with the same method. You just need to measure the control sample in the same way and then use it to measure the ration of your protein to control

[@carlinlapo]

8 minutes to only mention that the line should touch wtf

[@AliceritaE]

You're welcome!

[@AliceritaE]

If you have gone through the comment section, you would have seen that it's more than just drawing a single line. But to each one their own 😏

[@carlinlapo]

@@AliceritaE I just meant that I expected more troubleshooting according to the title, a bit clickbaity hahaha but nice video

[@AliceritaE]

@carlinlapo you're the first to call the video a click bait. The video tutorial cannot accommodate everyone's curiosity. It provides the basic into gel quantification. If you're working with western blot gel, you need further steps and which you can do by yourself if you have the equation for the quantification.

[@carlinlapo]

@@AliceritaE that's what genius do, be the first one to discover something !! 😀