What transfer conditions should I use to blot a membrane protein of size 82kDa?

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What kind of post translational modifications can cause a huge change in the molecular weight of a protein with the exception of glycosylation?

I have run a 2D gel to identify certain protein isoforms in rat urine. I did a western with a antibody that binds to all isoforms of the protein. The expected size is around 17kD-20kD., however...

07 August 2014 5,984 7 View

How do I remove phosphate group from a protein that has been phosphorylated on its Histidine residue?

I wanted to know if there is a phosphatase that is specifically used for removing phosphate groups from histidine residue of my phosphorylated protein. Mostly, I have only heard of Ser/Thr...

07 August 2014 9,796 2 View

How can I solve my protein instability so it can be used in NMR and ITC studies?

I have a highly charged & low molecular weight bacterial protein that is highly unstable. I have tried purifying it with different tags including His tag, MBP tag, Thioredoxin tag, SUMO tag...

06 July 2014 3,653 14 View

What are the membrane transfer conditions I should use during western blot if i have two proteins of varying sizes?

If i need to do western blot of two proteins of varying sizes separated in the same gel (one 55kDa and one 13kDa), what should be the membrane transfer conditions that I need to use? Is 70V for 2...

06 July 2014 4,002 6 View

Does the use of Phos-Tag give a high background?

I will be incorporating Phos Tag in my protein gels to study histidine phosphorelay between two bacterial proteins. I wanted to know if there is a possibility of getting a high background upon...

06 July 2014 9,960 2 View

How to check protein stability?

I have purified my protein and I want to check its stability. If I keep a tube with my purified protein at 4C for a day, can I check its stability at the end by spinning it down and measuring OD...

06 July 2014 7,881 9 View

Does anyone know what might be causing the smeared bands on my western blot?

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

Can I use a HisTRAP column for affinity chromatography?

I'm working on selecting antibodies against a recombinant protein that has a His-tag. My idea is to first bind the recombinant protein to a HisTRAP column and then use this column for an affinity...

07 August 2024 505 3 View

Weak DAPI staining after immunohistochemistry - how to improve?

After immunohistochemistry of previously fixed in PFA and EtOH and then frozen 20 μm sections of zebrafish brain, DAPI staining is very weak (right) compared to the same sections stained without...

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How to isolate lymphocytes from mouse spleen?

I have tried several times to isolate lymphocytes from mouse spleen, but all attempts have been unsuccessful. I tried most available protocols. I used different dissociation media (HBSS with Ca...

04 August 2024 9,913 7 View

How to Add Missing Water Molecules in Protein-Membrane Simulations?

I have protein-membrane simulations (PDB, PSF, DCD) and have noticed that water molecules near the protein are not visible in the simulations. How can I fix this issue? Is there a way to place the...

04 August 2024 1,200 2 View

What is the best blank for nanodrop if I want to read a recombinant protein concentration?

Is it the "elution buffer" or the "dialysis buffer"? Note: I'll be using NanoDrop OneC

01 August 2024 967 3 View

Can i use the protease inhibitors during cell membrane vesicle preparation ？?

I am currently working on a project that involves extracting cell membranes, for which we disrupt the cells using sonication. During the initial extraction process, we add protease inhibitors to...

30 July 2024 7,077 1 View

What is the reason I get 400nm nanoparticles using an extruder with 200 nm membrane for cell membrane coating?

I am trying to coat my micelles and PLGA based nanoparticles with cell membrane using an Avanti mini extruder according to literature . I use 200nm membrane but my particles end up to be 400nm And...

30 July 2024 3,294 3 View

Suggestions for the Annexin V Positive Control ?

Hello everyone, I am currently using washed human platelets to stain Annexin V as a procoagulant marker. Additionally, I am staining with PerCP-CD61 to identify platelet cells. So far, I have...

29 July 2024 8,624 1 View

Do some Staphylococcus aureus strains have in vitro antimycobacterial activity ?

Some Staphylococcus aureus strains Inhibit the growth of Mycobacteria in Mueller Hinton Agar medium containing 10% OADC. Do some Staphylococcus aureus strains have in vitro antimycobacterial activity?

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