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Questions related to Protein Purification Techniques
I have tired to purified it using protein A column (low salt) . However when i performed immunofluorescence, the purified antibody could only stain the peripheral and surrounding cytoplasm but not...
12 December 2016 510 6 View
Hi..I have over-expressed a protein (tagged with His) via retroviral transduction method in THP-1 cells. Now I am interested to check if there is any binding partner with my tagged protein in a...
12 December 2016 2,784 4 View
Hello, I'm currently trying to purify a his-tagged protein with AC. However, when running the HPLC I have a very dissapointing protein yield (both total protein and the protein of...
12 December 2016 7,435 7 View
Hi everyone, I am working on brain inflammation and I try to separate the mouse brain vessels from the parenchyma after the removal of the olfactory bulbs and the meninges. Currently I proceed to...
12 December 2016 9,018 2 View
I am purifying alpha synuclein protein, in that supernatant is dialysed against tris buffer. to avoid this step what is the alternative of this step?
12 December 2016 3,556 3 View
Hello all, I'm trying to determine the ubiquitin linkage site of a polyubiquitinated protein using HA-tagged ubiquitin mutants (K0, K48, K63, K48R and K63R). For this I'm immunoprecipitating my...
12 December 2016 4,303 0 View
Hi all,I recently ran a western blot using medial prefrontal cortex samples from PN17 rats. I followed the same protocol that other students have used in our lab (we prepare samples with RIPA...
11 November 2016 2,767 2 View
I am trying to purify a recombinant protein for its functional study and I want to do some biochemical assays. Due to hydrophobic nature of the target protein most of its fraction forming...
11 November 2016 5,165 4 View
Hi everyone, I'm looking for a working protocol for extracting proteins from formalin fixed brain tissue to be used for Western Blotting. I've tried a few protocols with little success and my...
11 November 2016 9,612 2 View
I extracted proteins from mouse thyroid tumors with RIPA buffer. Some lysates are red and I would like to remove hemoglobin befor protein quantification (BCA assay). How can I do it?
11 November 2016 5,076 7 View
while I am doing western blotting seems some polysaccharides are interfering with the interest protein and does not let the protein pass trough gel.I was wondering is there any protocol to...
11 November 2016 5,708 2 View
I have done a western blotting experiment using protein isolated from cultured cells to detect GAPDH. I got some faint bands. So I repeated the experiment with higher concentration of protein, but...
11 November 2016 1,660 11 View
I am using a T7 Megascript RNA kit from Invitrogen. I transcribe overnight at 37˚C, treat with DNase, phenol/chloroform extract, precipitate with isopropanol, then denature 30mins at 70˚C and let...
11 November 2016 5,275 3 View
My protein extraction methodology from bacteria produces a light brown pellet. I have been toldthat these colours or pigmens can be problematic for the downstram mass-spec analysis on the Q...
11 November 2016 1,661 6 View
I work on lentil by using rapd pcr. the first band marker. second band sample 3. Band didn't appear 4. Band sample. All of them same pcr condition. do you have any opinion? can I analyze in this form.
11 November 2016 8,132 7 View
I did not observe any smearing in PCR product on gel before digestion while after digestion appeared PCR product as a smear on agarose gel. Please suggest where I am going wrong or which reagent...
11 November 2016 1,442 4 View
Hi, I am trying to do a Western Blot (Akt1, phospho Akt1, Akt2, phospho Akt2) from HepG2 (human liver cancer cell line) proteins stimulated with insulin. Akt1 is detected, but not the other Akt...
11 November 2016 3,698 4 View
I am working on a thermophilic protein (pI: 7.72) purified using Ni-NTA chromatography (Final buffer: 25mM Pipes (6.6), 300mM NaCl, 1mM PMSF, 10% glycerol, 200mM imidazole). But the protein...
10 October 2016 8,348 24 View
A hybrid kinase protein which functions by phosphorelying the phosphate in His-Asp-His-Asp manner with second His being the HPT domain and second Asp being that of the response regulator. will...
10 October 2016 4,943 0 View
I want to study more about immune system of Mollusk, because they do not have clotting property in their body circulation when they get injured or wounded.
10 October 2016 2,459 1 View
I'm performing IMAC to purify my his-tagged protein (enzyme) from e. coli lysate. It seems I can get a good amount of my protein of interest but the problem is that I also get alot of coeluates,...
10 October 2016 7,103 7 View
I performed quick change site directed mutagenesis but I am not getting mutated DNA my primers are exact complementary with each other and purification grade is desalted, not PAGE/ FPLC purified....
10 October 2016 8,024 10 View
Maybe comparison of benefit,advantages, disadvantages of column HiTrap and Hi screen. Hope anyone can help. Thanks
10 October 2016 5,089 3 View
1. Spot 2 µl of samples onto the nitrocellulose membrane; 2. Let the membrane dry; 3.Block non-specific sites by soaking in 5% milk in TBS (0.5-1 hr, RT). Not adding tween-20; 4.Incubate with...
10 October 2016 4,678 7 View