After affinity through amylose and TEV cleavage, the protein is aggregated in GPC so I am not getting any active protein out of this. How can I minimise aggregation.
Hi, I deal with synthetases which are dimeric. SHould I consider their dimeric mol wt before calculating their molarity or should I go with the usual SDS PAGE mol wt which is monomeric in Kilodaltons
11 December 2018 3,369 0 View
And am using 1:400 dilution for my protein's antibody. With overnight 4 deg binding. Should I make it 1 hour at RT. I increased tween in PBS from 0.01 to 0.05 % . I got some less background. But...
09 October 2017 1,327 0 View
I am doing it with the sigma GST assay kit. My blank buffer consisting of PBS, GSH and cDNB is also giving an absorbance. So can you just give me a detailed description in case am making a mistake.
07 August 2017 8,973 2 View
I was inducing with 0.8 mM IPTG and kept 16-18 hrs post induction at 25 degree..am trying to reduce the temp and check the expression ; also lowering the salt and increasing imidazole...
06 July 2017 8,924 2 View
Am getting a chaperone at around 63 kD . I have my construct in pETM41. Am purifying with amylose resin and getting a chaperone after affinity. Will HIC after first step clean my protein?
06 July 2017 8,910 5 View
I got these smeared bands quite often lately. We typically run the gel at 140V with a 10-12% gel and do a wet transfer at 220 mA for 1.5 hr in cold room. We also noticed some dirty spots/dots (see...
10 August 2024 7,480 3 View
Hi! So i attempted to understand a novel protein behavior towards heat application by analyzing its secondary structure change. I subjected the protein to a thermal denaturation analysis using...
06 August 2024 1,989 3 View
I have been working on Red blood cell-derived extracellular vesicles as Antisense Oligonucleotide (ASO) carriers. We normally run agarose gel to quantify the loading efficiency. I used naked ASO...
06 August 2024 3,130 2 View
I am having an issue with my gel image where my PCR product is not appearing very bright on the gel. When I perform gel extraction, the A260/280 purity value is very low. I used the Qiagen gel...
05 August 2024 9,798 3 View
Dear All, I am trying to transfect a pCDNA3.1 vector containing my gene of interest. The purpose is to figure out the localization of the protein of interest. I have fused the protein with GFP on...
31 July 2024 9,892 4 View
Hello everyone, I performed a PCR yesterday, and the results showed no bands on the gel. Of course, I probably missed some crucial steps, like adding my samples to the PCR strips themselves, for...
31 July 2024 2,406 6 View
We are working on biopolymeric hydrogels. Our system is highly viscous and sticky, and the gel formed are high in strength. We are unable to use pH electrode and pH strip. Please suggest an easy...
30 July 2024 942 2 View
I am using CuBr/THPTA for a click reaction in total cell lysates. I am facing issues with my protein sample in non-reducing SDS-PAGE where it's not migrating properly and most of it remains at the...
29 July 2024 950 4 View
Hello , I established a stable cell line expressing GFP tagged to a centrosomal gene having G418 drug selection marker. I validated the stable line by IFA and Western blotting, results are fine....
29 July 2024 5,007 0 View
I want to do 2,3-butanediol dehydrogenase(BDH) enzyme purification to confirm its activity for 2,3-butanediol. Before that, I need to confirm which N or C terminal tagging is better for enzyme...
28 July 2024 366 3 View