SDS-PAGE

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Questions related to SDS-PAGE

Hi - I wanna extract my protein from priplasmic space of E.coli (DE3).do you have any functional method for it?

I have used smotic shocked method combined with TCA perception method but I cant see any result in SDS page ... so is that any other method that is more effective and I can use it as a substituted ?

09 January 2022 3,334 0 View

How to perform quantitative elution of proteins from BN-PAGE?

Good day! I need to elute proteins from polyacrylamide gel slices after BN-PAGE electrophoresis. The porblem is that the gel slices are relatively big - all the lane fragment upper from 1000 kDa...

27 December 2021 7,548 4 View

Want to Know where my staining and destaining procss went wrong?

My TA for an SDS-PAGE lab stained our gels for us and ended up with the following result. He used Coomassie blue for 30 min and then he left it overnight in an unknown destaining solution. Does...

10 December 2021 3,182 4 View

Should annealed oligos (20bp) be restriction digested before inserting into cut px330?

I'm using px330 plasmid, cutting it with BbsI, and using annealed oligos that are 20 bp in length. I've used RE with cutsmart buffer. Purified it used a qiagen gel purification column. For the...

10 December 2021 2,076 4 View

Why I cannot see clear proteins bands of Staphylococcus aureus with SDS page?

I am analysing the internal proteins of Staphylococcus aureus. I tried three different methods for extraction of the proteins prior loading on SDS page. I am growing the bacteria to log phase 0.4...

07 December 2021 2,034 5 View

NiNTA elution, experience with higher (>500mM) imidazole concentrations?

Which imidazole concentration do you use for protein- NiNTA elution? We use 300 mM, but i have seen protocols using 500 mM. Wouldn´t it be better, in theory, to use higher (>1/2 M)...

07 December 2021 7,432 3 View

Does centrifugation temperature affect agglomeration of AuNPs synthesized using the citrate method?

Hi! I prepared AuNPs using the citrate method and tried purifying them. I aliquoted the resulted AuNP solution in 2 mL volumes and centrifuged at 6000 rpm for 1 hr at 4 celcius as the first...

02 December 2021 3,605 9 View

What components in DSM cause sporulation of Bacillus subtilis?

For a practical course, I have to give an introduction into endospore formation in B. subtilis. We use DSM (Difco Sporulation Medium) to induce sporulation. As I am no expert in this field, I was...

01 December 2021 6,922 0 View

How to study of novel proteins in bacterial host?

Hi, I have validated some novel proteins in my targeted bacteria. Now, I want to study the effects of these candidates by overexpressing them inside the bacterium. I have targeted three phenotypic...

01 December 2021 6,665 1 View

Is that possible to detect a 3 kDa peptide using SDS-PAGE without tricine?

We are working with a recombinant peptide of 3 kDa, but we do not have tricine in our lab, only glycine. Is that possible to detect this peptide without using tricine in the SDS-PAGE? High...

30 November 2021 7,074 4 View

Could anyone tell me what's wrong with my electroporesis band?

i used the denatured collagen sample which was treated by 100 temperature, collagenase with 50U and trypsin with 0.1% for 10 mins, and put 7ul denatured collagen into each well. SDS-PAGE was...

19 November 2021 146 5 View

What is affecting my bradford assay?

Currently, I am trying to do a Bradford Assay on my MS2 capsid protein that is fused with APOBEC1 and I am having very low absorbances for this protein. My Bradford Standards and MS2-APOBEC1...

18 November 2021 246 5 View

How can we solve our SDS PAGE problem?

Lately in our lab, some of the gels have problems like smear image or strange markers. We have changed all the components like SDS, Laemmli, SDS running buffer, marker, Acrylamide, sds tank (mini...

11 November 2021 5,477 3 View

Hello.. did any of you ever do a native PAGE and SDS PAGE for xylanase enzyme production from Trichoderma sp.?

Hello.. did any of you ever do a native PAGE and SDS PAGE for xylanase enzyme production from Trichoderma sp.. I need a procedure plz

09 November 2021 6,708 2 View

How to unfold Protein using a ANTI-FLAG M2 Affinity Gel?

I am currently using the Anti-Flag Affinity Gel to pull down a Flag protein inserted on the c-terminal of a specific protein. I have been unable to detect the flag protein via SDS page. Recently,...

01 November 2021 8,704 3 View

How can I make my SDS PAGE bands look better?

I use 8% gel And 5% stacking gel. I use BSA protein solution diluted into PBS for different concentrations. I ran it for 45 mins at 130V. However, my bands look chubby. How can I fix this? Could...

27 October 2021 8,761 3 View

Why is my sample smeared from top to bottom throughout the entire lane of my western blot?

Hello, Please see attached a snippet of my western blot for this inquiry. Some brief background on this particular sample ("1"): This sample is a Mycobacterium spp. cell culture that was...

26 October 2021 1,614 3 View

Have you got experience with open source ELNs?

Dears, I am a lab manager of a small group and we would like to shift from paper lab book to an electronic one. I found online the Indigo ELN software. Do you have experience with it or other...

26 October 2021 4,325 0 View

Can I detect purified Egg yolk IgY antibodies using 4 to 12 % linear gradient SDS PAGE gel?

My objective is to extract and purify certain antigen specific chicken egg yolk antibodies. I have found through literatures that, some of the researchers used linear gradient SDS gel to detect...

25 October 2021 6,602 3 View

Why is Fc tag only present on a fraction of my produced recombinant proteins?

Hi! I have trouble finding literature on this. Can someone please share some information on what could be the cause of a glycoprotein-Fc-tag fusion protein being expressed in HEK cells missing its...

20 October 2021 3,054 3 View

How can i get a good intensity bands in native page?

i am performing an native page of my protein but i am not getting good bands can any one help me to figure it out

09 October 2021 9,205 6 View

Why am I seeing uneven staining from silver stain?

I achieved uneven staining using silver stain on my SDS-PAGE gel, with my ladder (New England Biolabs #P7719S) staining the most, and a gradient down to my 5h timepoint staining least. To clarify,...

07 October 2021 3,333 4 View

Can any one please tell me, how to calculate the survival fraction?

Is there any formula, basing on that one can calculate the survival fraction in in vitro clonogenic assay? In cancer cells upon drug treatment, colony growth significantly reduced in comparison to...

06 October 2021 1,292 1 View

For gelatin in SDS PAGE analysis...i got smeard band why?

how to get clear band....please provide details....attached the image

26 September 2021 334 3 View

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