What can I understand from this SDS-PAGE? How do I know what the different bands are?

More Vanshika Singh's questions See All

Two pendulums with different mass bobs and different string lengths dropped from the same height. Time of fall?

05 June 2017 2,185 5 View

To perform a HIs-Tag Protein purification, what is the correct way to make the lysis and elution (Sodium Phosphate) buffer?

Can I bring the pH to 8 using NaOH? What are the pros and cons of that? Will it work if I add only sodium dihydrogen phosphate for the phosphate ions and skip the disodium hydrogen phosphate?

04 May 2017 7,386 5 View

Protein-aptamer gel electrophoresis help, please??

Hi, I have problems with running gel electrophoresis. I have tried agarose gel electrophoresis and native PAGE. I have two proteins, which have molecular weights of ~30kDa and ~180kDa and two...

03 March 2021 4,275 4 View

What are some important techniques for protein induction and SDS-PAGE analysis?

Hello everyone. I am fairly new in protein chemistry field. I am trying to perform a protein induction hoping to purify my protein of interest using FPLC method. However, I am having trouble on...

03 March 2021 8,237 3 View

SDS page software for analysis?

I did sds page to determine the difference in protein expression between 3 types of vaccine , but i don't have scanner or densitometer available. . so i wonder if there is a software to analyze...

02 March 2021 6,270 3 View

New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before?

Question to you and THEM, the New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before? There appears to be a core problem for...

02 March 2021 3,024 2 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

28 February 2021 4,521 3 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

28 February 2021 7,370 3 View

Dominique Liger

Hi there,

It would be easier to help you if you mention the content of each lane. From a general point of view you should load more material in order to get a clearer contrast between bands and gel background.

Mangal Singh

It seems that yours is a low molecular weight protein and the IMAC was succesful as the protein is concentrated and comparatively pure. You need to increase the percentage of acrylamide gel for sharper bands in the low molecular weight region and also please mention the content of each lane for further help.

Vanshika Singh

@Dominique- Thank You, sir. I will add more content next time to obtain clearer bands.

These are mutants of SUMO-1 protein, a ubiquitin like modifier. Order of loading, starting from right-

Marker - wash of Mutant 1- gradient elutes (50mM- 300MM imidazole) of mutant 1 in lanes 3,4,5.

The same is repeated for two other mutants in the same order.

What I am looking to understand is, what band represents what. Are those faint bands also giving me some information, or the blots at the end of the gel is the section that gives me the most information about the elution fractions.

@ Mangal Singh. Thank you for the reply, I have added the details of the lanes above.

Could you help me understand how you reached those conclusions about the protein with this gel?

SUMO1 as i can see is a low molecular weight protein and with a hexahistidine tag may be 13 kDa or so. for this range you have to run tricine SDS PAGE for a sharp band. As i already mentioned, IMAC concentrates the hexahistidine tagged protein which is visible in the blurred big stained spot and also this is present only in the lanes where you applied Imidazole containing buffer.

Thanks Mangal for that informative answer. I was also wondering how to detect if some proteins have co-precipitated in those "blurred big stained spots" or are they just the SUMO-1 mutants.