Questions tagged [western-blot]
Questions pertaining to western blots, which are used for detecting specific proteins (sometimes called protein immunoblots).
56
questions
1
vote
0
answers
26
views
Does it make sense to calculate the phospho protein (serine) : phospho protein (threonine) of the same protein in western blot?
I want to indirectly measure the nuclear accumulation of DARPP protein through phosphorylation. Their nuclear transport is controlled by phosphorylation of Threonine 34 and Serine 97 residues.
S97 is ...
- 10.1k
2
votes
1
answer
1k
views
Can I use grayscale images when working with ImageJ?
I am using ImageJ to analyze Western Blots. I have scanned films in as grayscale images because this is how we did it in my old lab. People in my current lab are not satisfied with that explanation ...
- 10.1k
4
votes
1
answer
662
views
How to remove bad lanes in ImageJ Westernblot analysis
I use ImageJ to do an analysis of a Westernblot Image.
If everything goes as wanted the workflow is fine. But if I do something wrong creating a lane there is no undo for a lane and also no way to ...
- 10.1k
1
vote
0
answers
82
views
Can you store 5% BSA, in TBST, in -20C?
I have stock solution of 5% BSA prepared in TBST I use to make primary antibody dilutions for western blot. I'll admit I just assumed -20C, with freezing and thawing as needed, was an acceptable means ...
- 11
5
votes
1
answer
2k
views
Western blotting with multiple antibodies
Normally I wash/detect with one primary/secondary-HRP antibody pair, strip, then wash/detect with the other primary/secondary-HRP pair which works well. However, I recently started working with a HRP-...
1
vote
1
answer
185
views
choosing the right housekeeping gene for Western Blotting analysis for liver lysates
B- actin is used a great deal for quantitation of liver lysates. What other alternatives are there for WB analysis? should we opt for GAPDH or tubulin?
- 1,305
2
votes
1
answer
132
views
Does beta-actin have to be consistent in Western Blots?
I have been running Western Blots on rat brain tissues from rats that have been subject to neurological disorders.
I loaded the samples on the gel to ensure that for each tissue, I had a constant mass ...
- 12.1k
0
votes
1
answer
70
views
In enhanced chemiluminescence in western blots, will the horseradish peroxidase eventually get used up?
I am learning about enhanced chemiluminescence in Western blots. I have read online that in enhanced chemiluminescence that horseradish peroxidase (HRP) catalyses the oxidation of luminol to 3-...
- 226
1
vote
1
answer
251
views
Western blot trouble shooting - low voltage and yellow sponges during/after transfer
I have a western blot troubleshooting question that I haven't been able to find the answer to in manufacturer troubleshooting guides.
As a bit of background, I was transferring 2 western blots ...
- 41
0
votes
1
answer
41
views
Why is it good practice to prepare western blot samples in pairs when you have two experimental groups?
I have done some Western blots where I have analysed the expression levels of a certain protein in brain homogenates from wild-type and knockout mice. When preparing brain homogenates, I was advised ...
- 3,852
2
votes
0
answers
34
views
In phospho/pan analysis in Western blots, what is best way to normalise to an internal loading control?
I am analysing the expression of a protein kinase X that is a phosphoprotein through Western blots. I have labelled the membrane for both the phosphorylated form of the protein and also for the total ...
- 1,671
1
vote
1
answer
487
views
Can you use images taken at different exposure times in western blot image analysis?
I am doing Western blot data analysis where I have images from a number of experiments (where the samples in the experiments are biological/technical replicates). For each experiment, I labelled the ...
- 12.1k
0
votes
1
answer
289
views
When analysing phosphoproteins via Western blot, why is total protein level of the target protein recommended as an internal loading control?
I am analysing the expression of a protein kinase via Western blots, and it is a phosphoprotein. I have labelled my membrane with antibodies against the phosphorylated form of the protein (using ...
- 12.1k
1
vote
0
answers
90
views
Why do you need to calculate the lane normalisation factor when doing western blot data analysis?
I am learning about western blot data quantification from some online resources. I have read about methods to normalise the data.
I have seen in a number of resources, such as this handbook and this ...
- 1,671
2
votes
1
answer
175
views
Can Western Blots be used to quantify the activity of a protein?
I am new to Western Blot analysis and I have recently done my first two. I am studying a phosphoprotein (a protein kinase) that can be both activated and inactivated via phosphorylation at a specific ...
- 6,222
1
vote
1
answer
156
views
What is the best way from remove background signal from a band when doing Western blot image analysis?
I am doing image analysis of a Western Blot in Image J. I have calculated the total intensity of my protein bands of interest through outlining each band using the rectangle tool in ImageJ, and ...
- 10.1k
0
votes
1
answer
85
views
What is background signal in a Western blot?
I have done a western blot and I want to remove the background signal when doing densitometry analysis of my protein bands of interest. I have read some articles online such as this one regarding high ...
- 4,714
-1
votes
1
answer
117
views
In western blots, do loading controls have to be proteins that are in equal amounts in each lane?
I have recently done a Western Blot and I am doing data analysis on my blots. I am studying the protein GSK3, which is a phosphoprotein.
I have labelled my membrane against active GSK3 and inactive ...
- 544
4
votes
1
answer
700
views
What are the units of the band intensities in a western blot image?
I have done a Western blot and I am measuring the band intensities in Image J. Using the rectangle tool, I have outlined the band and then calculated the area and the mean intensity of the band. I ...
- 6,222
1
vote
1
answer
69
views
Is it necessary to calculate lane normalisation factor when doing western blot data analysis?
I have recently done my first western blot and I am doing data analysis to quantify my blot. I have labelled my membrane against inactive GSK3 and active GSK3 which are phosphoproteins so I am using ...
- 226
0
votes
1
answer
634
views
What is meant by 4 –12% or 8% SDS-PAGE?
I am reading a journal paper and I am looking at the materials and methods section. Regarding the Western blot method in the paper, I have come across the following statement:
Proteins were separated ...
- 8,296
0
votes
1
answer
52
views
AMPK, PAN-AMPK, western blot
What does it mean by adding PAN before the AMPK. I am trying to do a western blot assay on AMPK and I am confused by PAN-AMPK, AMPK. does it mean pancreases? Is pan-ampk the total ampk?
CommunityBot
- 1
1
vote
1
answer
43
views
Need help interpreting Western blot data
So the paper I am reading (here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094582/) uses densitometric units to quantify the western blots.
The authors mentioned that the unphosphorylated proteins ...
- 10.1k
1
vote
0
answers
47
views
Problem with Caspr antibody in western blotting
I have been working on optimizing western blotting for Caspr (contactin associated protein), but I am having difficulties and would like suggestions.
I ran my proteins on a 40% acrylamide gel,
I ...
- 21
0
votes
2
answers
927
views
Preparing sample for SDS PAGE
I have more than 10 cell lysate samples (70 µL each) whose concentration varies from 1.9 mg/mL to 4.8 mg/mL. I have 5X and 2X SDS sample buffers. I would like to prepare SDS PAGE samples in such a way ...
- 35.6k
0
votes
1
answer
380
views
What is the role of triton in buffer for western blot assay?
What is the role of triton in buffer for western blot assay?
I understand it has to do with preventing non-specific binding of the antibody.
CommunityBot
- 1
3
votes
2
answers
31k
views
What is the role of glycine in the running buffer for SDS-polyacrylamide gel electrophoresis
We used pH6.8 in stacking and pH8.8 in resolving gel. In the class, the professor explained that the glycine change is like:
...
3
votes
1
answer
320
views
Western blot transfer issues
I am having some issues with transfer efficiency to nitrocellulose membranes. To get all the technical info out of the way:
Transfer method: Wet, tank transfer overnight at 30V with normal tris-...
CommunityBot
- 1
5
votes
1
answer
17k
views
How to prepare sample for SDS PAGE?
I usually take my protein sample 0.8ml and sample buffer(2X) 0.2ml for my sample preparation in SDS PAGE. Am I using correct proportion? my protein sample concentration is 4.1mg/ml. What is the ...
- 1
5
votes
2
answers
2k
views
High Current (Speed) Transfer Buffer Recipe
Does anyone know an effective buffer mix to use for high current Western transfers? We are successfully using the vendor's premixed buffer to transfer a wide range of protein sizes to PVDF membranes ...
CommunityBot
- 1
2
votes
1
answer
21
views
What for a Kinase Assay? [closed]
I am wondering why to use a kinase assay, since we can extract the proteins from cells and then do a Western with the specific antibodies we want to use.
- 15.3k
8
votes
1
answer
552
views
Optimal pH of protein buffer? Basic principles to adjust buffers according method and analysis
Protein buffers such as PBST, which is used in western blotting, are normally adjusted to pH 7.4. When I try to find why, I find some information about optimal pKa for protein stability. Im not sure I ...
- 710
1
vote
1
answer
4k
views
PBST vs. TBST buffer in western blotting
What is the advantages and disadvantages of using either PBST or TBST in western blotting, or while working with proteins in general? Are there other buffers which are also used for western blotting, ...
- 15.3k
1
vote
1
answer
114
views
Use of western blotting
I ran across this paper recently.
http://www.ncbi.nlm.nih.gov/pubmed/21151122
I am confused on their use of Western blotting. Their unmodified protein (no PTM group) has a band and their modified ...
- 131
1
vote
0
answers
93
views
Using only one antibody to detect COX-2 enzyme
If I wanted to detect COX-2 from a western blot test, would only a primary antibody work? I'm on a budget so does anyone know of relatively cheap COX-2 antibodies?
- 16.1k
2
votes
2
answers
9k
views
Why is beta actin commonly used as a control in Western blots?
It is about the western blot question. The paper:http://www.sciencedirect.com/science/article/pii/S1931312813001145.
In the western blot experiment in this paper, they use beta actin as control. And ...
- 2,377
4
votes
1
answer
453
views
What equation to compare protein isoforms in a Western Blot?
The protein isoforms I am interested in comparing appear as distinct bands on the gel I have already run. I have an Excel sheet with optical density measurements I obtained using ImageJ; it looks ...
- 35.6k
4
votes
0
answers
327
views
Relative densitometry from SDS PAGE
I'd like to perform densitometry on a Coomassie stained SDS PAGE gel to compare a recombinant protein's expression levels under two conditions. I'm using BioRad's Image Lab software.
My questions are ...
- 103
0
votes
1
answer
68
views
Molecular weight of my 2-D gel
I am a little confuse when I try to figure out the molecular weight of the marker on my gel. I used NuPage Novex 4-12% bis-tris gel and Mark12™ Unstained Standard as a marker. Could please someone ...
- 17.7k
2
votes
1
answer
148
views
Why does my anti-ubiquitin antibody visualization not work on my PAGE gel?
I am using 2D gel electrophoresis to visualize polyubiquitinated proteins. However, while I can see actin and heat shock protein using when appropriate antibodies, I cannot visualize them using anti-...
- 1,116
3
votes
3
answers
2k
views
LCMS/MS versus Western Blot
I have a general question regarding which method would you recommend me to use if I would like to investigate the difference in the level of several proteins in tissue samples and compare different ...
- 53
5
votes
2
answers
4k
views
Why proteins are not visible on my membrane after ponceau staining?
I have a problem in western blot that I can't resolve by myself.
When I am use to add 100 microgram of proteins for each sample but after running and transfer its impossible to me to see bands of ...
- 123
5
votes
1
answer
2k
views
Are there any disadvantages to HRP conjugated PRIMARY antibodies
I need to detect a FLAG tagged protein on a western blot and have to order an antibody. I'm not expecting to see a massive amount of my protein. To keep analysis time to a minimum I am considering a ...
- 51.6k
4
votes
0
answers
88
views
Procedure for doing western blot [closed]
I am writing a step by step guide for doing a western blot for a class. It is intended for any one with basic Biology lab skills. I am hoping people will review my draft and give feedback on how to ...
- 41
2
votes
0
answers
213
views
Why HIS tag is not detected in WB or dot blot?
I would like to know why the recombinant protein with his tag at c terminus is not being detected neither in western blot or dot blot.
I'm trying to validate protein expression of four proteins. ...
- 971
4
votes
3
answers
473
views
Western Blot: Fischer ECL substrate 2:1 instead of 1:1?
i recently developed a b-actin-ab-incubated PVDF-WB-Membrane with the Thermo Scientific Pierce ECL Kit. According to the manufacterers orders i mixed Reagent 1 and Reagent 2 1:1. But unfortunately i ...
- 21
10
votes
1
answer
3k
views
Mass spectrometry versus western blotting for validation
I have mass spectrometry data (LC-MS/MS) from rat cortices under either drug or control treatments. The results were performed in triplicate (three pairs of rats, drug or control per pair). In ...
- 345
3
votes
1
answer
948
views
Western blot alternatives
Given an in vivo sample from an experimental infection, I would like to see if a bacterial protein is present.
I've been thinking about Western Blot using sera from animals infected with the bacteria....
- 3,079
2
votes
1
answer
235
views
can a bacterial protease inhibitor cocktail be used for Western Blotting involving HUVEC cells and HT-29 adenocarcinoma cells?
Dear fellow biochemists,
I need some advice on Western Blotting, more specifically the use of certain protease inhibitors with the RIPA cell lysis buffer and protease inhibitor cocktail. A Millipore ...
CommunityBot
- 1
4
votes
2
answers
192
views
Is there a strong reason to be sceptical about the "cured HIV patient" being reported by mainstream media?
There's a story going round the news about a baby that was, apparently, cured of HIV using a cocktail of drugs at an early age. The story piqued my interest, but details seem scarce. One of the main ...
CommunityBot
- 1