Hi there, I was wondering if it is possible to calculate the fluorescence quantity absorbed from SDS-PAGE gels? My goal is to create a graph that represents the Molecular weight of the band on the x-axis and the volume of fluorescence detected along the y-axis. Thanks.
hello, thanks for reaching out. I am sure the experimental design you planned is definitely doable. once i worked with a coomassie blue staining of proteins in an SDS-PAGE gel. I snapped a picture of this gel using a gel documentation machine. my experiment was to compare the protein expression in the control vs treated cells and study the differential protein expression. I used ImageJ to quantify the coomassie blue staining intensity using the same method like quantifying western blot bands. The end result was to select the differentially expressing proteins according to their molecular weight for further experiments. I have a separate video on western blot band quantification (fluorescence). Here is the link to the video (th-cam.com/video/u-u3G7JhIAo/w-d-xo.html). let me know if you need more info.
Hello, thank you so much. If I got you correct, you are referring to the original gel image before background subtraction. Then I think I need to thank our existing protocol in the lab.
Hello, thanks for the question. In this video the default unit for both area and percent area is pixel. The unit of area is pixel square. Pixels can also be converted to any unit of your choice such as mm, μm etc. If there is a scale bar in the image, then a specific unit can be assigned. Hope this helps.
Why are we dividing it with gapdh instead of subtracting? Also, how to compare the results of untreated and treated cells when GAPDH value varies. please reply
Thanks for the question Sonam. Here is an article you might like to look at link.springer.com/protocol/10.1007/978-1-0716-1514-0_7 In this article (densitometry) the calculation for semi-quantitative RT-PCR relative gene expression is shown as gene/B-actin. you may find this at 3.4 line 5. There are other housekeeping genes like B-actin, 18S rRNA, HPRT1... I think i would select a housekeeping gene that may not vary with the treatment. Hope this helps.
Hello, thanks for reaching out to @nrtTAYE. We use the percent area values to calculate the results by normalizing to housekeeping genes like GAPDH. i follow this method for my publications.
Hello, there are two ways to import the jpeg image into this software. 1) just drag and drop the image directly into the software, and 2) click on file, open, select the jpeg image and click open. Hope your query is answered.
Hi sir, at the end of the video, you had mentioned about using the values to plot the graph, can you show me what kind of plot and how can it able to determine the densitometric analysis of the agarose gel band, Thank you
Hello, thanks for reaching out to @nrtTAYE. once the percent areas are obtained and normalized, the fold changes/normalized values can be used to plot a graph. Please have a look at this video tutorial th-cam.com/video/oIarw23GIw4/w-d-xo.html you may plot either a scatter or a bar graph using any software like excel, sigma plot, origin or graph pad to show the desitometric differences between the agarose gel bands.
hello, it is possible to quantify the spots from a TLC, it is similar to analyzing dot blots. I will upload a video tutorial on dot blots next week. if this is an urgent matter please drop your email and I can get back to you with the step by step process to analyze.
Hello, the video for dot blot quantification is available online now at th-cam.com/video/U1l5jA3DL0s/w-d-xo.html I believe this method will apply for TLC spots too. All the best.
Great tutorial!
Thank you very much for your warm words! I'm delighted to hear that you find interest in my tutorial videos.
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Thanks ritu
Hi there, I was wondering if it is possible to calculate the fluorescence quantity absorbed from SDS-PAGE gels? My goal is to create a graph that represents the Molecular weight of the band on the x-axis and the volume of fluorescence detected along the y-axis.
Thanks.
hello, thanks for reaching out. I am sure the experimental design you planned is definitely doable. once i worked with a coomassie blue staining of proteins in an SDS-PAGE gel. I snapped a picture of this gel using a gel documentation machine. my experiment was to compare the protein expression in the control vs treated cells and study the differential protein expression. I used ImageJ to quantify the coomassie blue staining intensity using the same method like quantifying western blot bands. The end result was to select the differentially expressing proteins according to their molecular weight for further experiments. I have a separate video on western blot band quantification (fluorescence). Here is the link to the video (th-cam.com/video/u-u3G7JhIAo/w-d-xo.html). let me know if you need more info.
Thank you!!
Welcome, hope you found it useful
@@nrttaye4033 Totally!
Me salvaste hoy jajaja ❤
Gracias. glad you found it useful
How did you manage to make the image of the comet so clear?
Hello, thank you so much. If I got you correct, you are referring to the original gel image before background subtraction. Then I think I need to thank our existing protocol in the lab.
Thankyou.. anyway what unit do you use for each number of the results of the gene expression quantification?
I recently check some paper use nanogram, is that same for quantification using imagej?
Hello, thanks for the question. In this video the default unit for both area and percent area is pixel. The unit of area is pixel square. Pixels can also be converted to any unit of your choice such as mm, μm etc. If there is a scale bar in the image, then a specific unit can be assigned. Hope this helps.
5:00, what would the graph be like in terms of x and y axis, and units
y axis will be the fold change of the gene expression and x axis will be the names of the test or conditions
Why are we dividing it with gapdh instead of subtracting? Also, how to compare the results of untreated and treated cells when GAPDH value varies. please reply
Thanks for the question Sonam. Here is an article you might like to look at link.springer.com/protocol/10.1007/978-1-0716-1514-0_7
In this article (densitometry) the calculation for semi-quantitative RT-PCR relative gene expression is shown as gene/B-actin. you may find this at 3.4 line 5.
There are other housekeeping genes like B-actin, 18S rRNA, HPRT1... I think i would select a housekeeping gene that may not vary with the treatment. Hope this helps.
Hi, using which value do you calculate for significance of results? Should we use normalized values to GAPDH or original values?
Hello, thanks for reaching out to @nrtTAYE. We use the percent area values to calculate the results by normalizing to housekeeping genes like GAPDH. i follow this method for my publications.
I'm not able to copy the jpeg image into this software
Hello, there are two ways to import the jpeg image into this software. 1) just drag and drop the image directly into the software, and 2) click on file, open, select the jpeg image and click open. Hope your query is answered.
Hi sir, at the end of the video, you had mentioned about using the values to plot the graph, can you show me what kind of plot and how can it able to determine the densitometric analysis of the agarose gel band, Thank you
Hello, thanks for reaching out to @nrtTAYE. once the percent areas are obtained and normalized, the fold changes/normalized values can be used to plot a graph. Please have a look at this video tutorial th-cam.com/video/oIarw23GIw4/w-d-xo.html you may plot either a scatter or a bar graph using any software like excel, sigma plot, origin or graph pad to show the desitometric differences between the agarose gel bands.
Can u do this to analyze spots in a TLC?
hello, it is possible to quantify the spots from a TLC, it is similar to analyzing dot blots. I will upload a video tutorial on dot blots next week. if this is an urgent matter please drop your email and I can get back to you with the step by step process to analyze.
Hello, the video for dot blot quantification is available online now at th-cam.com/video/U1l5jA3DL0s/w-d-xo.html I believe this method will apply for TLC spots too. All the best.
@@nrttaye4033 thank you!! Have a nice day good sir
@@OlgaT1498 you are welcome. Thank you