High background on my Phos-tag gel western blot?

More Stella Wigglesworth - Littlewood's questions See All

Which is the appropriate statistic test to examine the relation between students' affective state and academic performance?

Which test should I use to compare students' mood effects on academic performance? I want to compare mood measured on a likert scale (from point 1= very sad to point= 5 very happy) to three...

04 February 2021 1,907 5 View

How can you derive a and n parameters of the Van Genuchten equation for horticultural mediums?

Dear all, I am trying to figure out how to derive and n parameters of the Van Genuchten equation for compost samples taken from an urban farm. RETC has predefined values for these parameters for...

26 January 2020 7,843 3 View

I am trying to form microspheres with PCL and PVA using a mechanical stirrer but the polymers keeps forming clumps. What do i do to disperse them?

I prepared a 1% PVA, and dissolved 500 mg of PCL in 5 ml of DCM. Then I introduced the PCL solution into PVA while stirring at 2000 rpm for 2 hours, after which i place it on magnetic stirrer to...

15 April 2019 7,177 4 View

Can normal colon cell lines form spheroids?

I am growing spheroids from HT29 colon cancer cells, and I need some sort of control cells to be compared with the HT29. Now, I should grow the control cell into spheroids and then use these new...

30 October 2018 3,719 12 View

Please, can I get a research paper or an article on lipid extraction from ogbono and egusi using chloroform-methanol method ?

I am working on a project that involves lipid extraction using chloroform-methanol as solvents. I washed the product with water-methanol and about to use a rotary evaporator for evaporation. But...

10 May 2018 4,089 3 View

What is the effect of differentating 3T3-L1 preadipocytes in differentiation cocktail for 3 versus 7 days?

I noticed that some protocols use differentiation media for 2-3 days while others go for 5-7 days before changing to maintenance media. Why is that? Is an adipocyte grown in DM 3 days + MM 4 days...

08 December 2017 1,792 1 View

How to calculate sample size for a moderation study?

Hi, I am doing a study design and planning to do a moderation study. This is the simplest model. However, I am struggling with the calculation of sample size. How do I calculate the sample size...

23 April 2017 5,449 8 View

I want to take for the first time CHO cells in culture. How can I get 100% sterile glass Erlenmeyer flasks to use them again in the cell culture?

29 November 2015 9,661 3 View

How do I design primers that will distinguish between highly homologous transcripts?

10 August 2014 4,948 4 View

How can I get my ligation to work?

I've been attempting to subclone an insert from one vector to another by PCR amplification with primers that incorporate XhoI/EcoRI sites at the ends of the insert and then digesting the insert...

30 July 2014 4,230 6 View

Protein-aptamer gel electrophoresis help, please??

Hi, I have problems with running gel electrophoresis. I have tried agarose gel electrophoresis and native PAGE. I have two proteins, which have molecular weights of ~30kDa and ~180kDa and two...

03 March 2021 4,275 4 View

What are some important techniques for protein induction and SDS-PAGE analysis?

Hello everyone. I am fairly new in protein chemistry field. I am trying to perform a protein induction hoping to purify my protein of interest using FPLC method. However, I am having trouble on...

03 March 2021 8,237 3 View

SDS page software for analysis?

I did sds page to determine the difference in protein expression between 3 types of vaccine , but i don't have scanner or densitometer available. . so i wonder if there is a software to analyze...

02 March 2021 6,270 3 View

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

28 February 2021 7,370 3 View

How can I represent SDS-PAGE results for machine learning analysis?

I am trying to classify and analyze the results of an SDS-PAGE based array for bacterial detection using machine learning, but I have trouble finding the best way to represent the results with...

27 February 2021 9,176 3 View

Will a 10xHis-tag prevent anti-MBP from binding to MBP?

I have a protein with a 10xHis-MBP tag on the N-terminus. I'm fairly certain I am expressing my protein, but I'm not getting a signal on my western blot. Is there a possibility that the 10xHis tag...

25 February 2021 8,404 3 View

Why my agarose gel staining sometimes looks not homogenic but like gradient (see the figure)?

I am worried about this overexposure of the upper part of the gel in the picture. Is it possible that this is the result of too much concentration of ethidium bromide? Why is there such a big...

25 February 2021 8,140 3 View

Why tick's salivary gland extract didn't run in the gel (SDS PAGE )??

Greetings, I want to make SDS PAGE for my sample (Tick salivery gland extract), I tried concentrations 10%, 12%. I also tried high molecular weight ladder. But the protein stopped at the top and...

25 February 2021 7,698 3 View

EMSA troubleshooting. Protein-DNA complex not migrating, cold probe lane with multiple bands. Can anyone help?

I'm running an EMSA to show the DNA binding activity of recombinant proteins versus the Wild type. Previous assays run by my research group using a different test system show the protein-DNA...

24 February 2021 8,325 1 View

Aleksandr Kalashnikov

High background on Phos-tag gel western blots can be a common issue. Here are some suggestions to reduce the background and improve the signal-to-noise ratio:

Use a lower concentration of Phos-tag: If you're using a high concentration of Phos-tag, try reducing it to minimize background noise.

Optimize the transfer conditions: Ensure proper transfer of proteins from the gel to the membrane. You may need to adjust transfer time, voltage, or buffer composition. Also, consider using a lower percentage of methanol in the transfer buffer.

Prolong the blocking step: Blocking the membrane for a longer duration (e.g., overnight) can help reduce non-specific binding and background.

Increase washing steps: Increase the number and duration of washing steps with TBST (Tris-buffered saline with Tween 20) or PBST (Phosphate-buffered saline with Tween 20) after primary and secondary antibody incubations.

Use appropriate primary and secondary antibodies: Ensure the antibodies are specific to the target protein and compatible with the detection system. Verify their concentrations and incubation times as per the manufacturer's recommendations.

Change detection method: If you are using chemiluminescence (ECL), you might consider trying an alternative detection method, such as fluorescence-based detection, which can offer a better signal-to-noise ratio.

Optimize imaging conditions: Adjust exposure time and settings of the imaging system to ensure the best possible signal-to-noise ratio.

Check the quality of reagents and solutions: Ensure that all reagents and solutions used are of high quality and free of contaminants. Replace old or expired reagents.

Satyendra Mondal

Dear Stella Wigglesworth - Littlewood

I have experienced this problem with Phospho-blot method. Lower your total protein load per lane. When there is too much protein loaded, the white band appears.

If you are using wet transfer method, use 0.1% SDS in the transfer buffer. It helps lowering the background. I hope it will help.

Best regards.

Anjali Srivastava

If you are observing a high background on your Phos-tag gel Western blot, there are several potential causes to consider. Here are a few possibilities:

Non-specific antibody binding: The primary antibody used to detect your protein of interest may be binding non-specifically to other proteins or cellular components present in your sample, leading to high background signal. This can be addressed by optimizing the antibody concentration and incubation time, and by including appropriate blocking steps in your protocol.

Incomplete blocking: If the membrane is not blocked sufficiently, non-specific binding of the primary antibody can occur. Ensure that you are using a blocking solution such as 5% non-fat dry milk or 3% BSA and blocking for an appropriate amount of time.

High protein concentration: If your sample contains high levels of protein, it can contribute to non-specific binding of the primary antibody, leading to increased background signal. Diluting your sample appropriately may help alleviate this issue.

Incomplete wash steps: Incomplete washing of the membrane can also lead to high background signal. Ensure that you are washing the membrane thoroughly with the appropriate wash buffer, and that you are using fresh buffer for each wash step.

Cross-reactivity with Phos-tag reagent: The Phos-tag reagent may cross-react with other phosphoproteins in your sample, leading to non-specific signal. It is important to include appropriate controls, such as a non-phosphorylated protein, to confirm that the signal you are detecting is specific to your protein of interest.

By carefully considering these potential issues and optimizing your protocol, you should be able to reduce the background signal on your Phos-tag gel Western blot and improve the specificity of your results.