Why my Gibson assembly doesn't work?

I'm assembling 3 PCR fragments with my vector pBAD and transforming them into dh5a. The second PCR fragment has 4 variations to it and the template for this PCR fragment comes from a plasmid. I've...

01 February 2019 5,696 3 View

Does anybody know where to purchase anti-H7 for flagellin in escherichia coli?

Im specifically looking for an anti-mouse antibody for flagellin (antisera H7). I've checked papers and googled extensively but couldnt find it. Any ideas?

03 April 2017 2,165 2 View

Does anyone have a good protocol for flagella preparation/purification?

I need a protocol to shear flagella off E.coli O157:H7. I have been using a protocol online that is basically resuspending an overnight culture in a syringe and then, spinning it down to acquire...

02 March 2017 6,637 2 View

How does one crystallise a protein with the presence of TCEP as the reducing agent, and with it's co-factors (NAD+ and Fe2+)?

I have a protein which I know is stable with the presence of co-factors (NAD+ and Fe2+). Recently i have been trying to purify it with the TCEP as a reducing agent since my protein is known to...

10 November 2016 185 2 View

Why mbp-cleaved protein disappears after anionic exchange?

I am purifying a protein (47 kDa) that is fused onto an MBP (42 kDa). The PIs for both proteins are 6.48 and 4.9 respectively. After purifying it using a dextrose column, I have buckets of fused...

04 May 2016 8,945 8 View

How to find self-associating motifs in a protein sequence?

04 May 2016 3,195 3 View

Why did the cleaving of my MBP tag on my protein failed?

I'm purifying a protein that is fused onto an MBP tag (42.5 kDa). My protein size is roughly the similar size of the tag (47kDa). Together, they form a complex about 89 kDa. After a run through a...

03 April 2016 8,667 3 View

How do I know if a flagella is in R or L conformation?

I have sequenced my sample and found some mutaions in the fliC gene. I have found that they have introduced some aa changes however, I would like to know if this changes the conformation into R or...

02 March 2016 7,471 1 View

How to generate GNOM file (.out) for DAMMIF runs?

I have .dat files from scatter that contain the Dmax of a protein and now I want to generate ab initio models. however I dont know how to generate .out files that are needed byy DAMMIF programs on...

09 October 2015 9,090 1 View

Why does the pressure alarm keeps going off during Nickel column purification?

I'm using a new column (5 ml Ni NTA HiFliQ column from Generon) to purify my protein AdhE which is Histagged and sized 96 kDa. However, during purification, the alarm pressure keeps going off even...

07 August 2015 5,183 10 View

Why does blocking with my immunogenic peptide cause nuclear fluorescence in ICC?

Hi all, I am validating a polyclonal antibody raised against human ANT4, through WB and ICC. In most of my controls, the antibody shows very little off-target binding, but when I pre-incubate the...

03 March 2021 5,999 3 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 View

Have someone successfully used the ER alpha (Santa Cruz sc-8002) antibody in immunostaining ?

Santa Cruz now recommends this new antibody (F-10) but I can't find a publication using this reference. I'm working on human et non human primate hypothalamus and so far, it doesn't work. Is...

02 March 2021 10,003 2 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Does anyone know an alternative product for cardiac Troponin T antibody for Immunofluorescence (Thermo # MA512960)?

We have been using Thermo Fisher anti-cardiac Troponin T antibody for immunofluorescence staining in FFPE mouse heart sections but it was recently discontinued. The literature shows it to be the...

01 March 2021 4,937 1 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

How does an antibody have full intact antibody + half antibody?

Hello, I was just wondering how does a antibody have full intact antibody + half antibody? Is it because of the drug conjugation?

28 February 2021 1,577 1 View

Does anyone use antinuclear antibody (ANA) and anti-Phospholipid antibody ELISA kit for Non Human Primate serum samples?

I am looking for anti-nuclear antibody (ANA) and anti-Phospholipid antibody ELISA kit for Non Human Primate serum samples. Any suggestions highly appreciated. Thank you.

28 February 2021 6,512 3 View

RIA. How to measure?

When you use RIA, with a control the unknown sample with antibodies, you bare in mind the binding sites of the antibodies. However you still need to measure labelled antigen (radioactive) and not...

28 February 2021 2,133 3 View

Anyone know of a good progesterone antibody?

I am looking for a good progesterone antibody to quantify levels of progesterone in zebrafish. Applications would be ELISA, and immunohistochemistry.

28 February 2021 5,929 2 View