# Large Protein Western Blot

**URL:** <https://scientistsolutions.discourse.group/t/large-protein-western-blot/873>\
**Category:** Western Blotting\
**Created:** [February 11, 2025, 11:04am UTC](https://scientistsolutions.discourse.group/t/large-protein-western-blot/873 "2025-02-11T11:04:47Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![Sarahcanon2025](https://avatars.discourse-cdn.com/v4/letter/s/f04885/32.png) [@Sarahcanon2025](https://scientistsolutions.discourse.group/u/Sarahcanon2025)\
**Post date:** [February 11, 2025, 11:04am UTC](https://scientistsolutions.discourse.group/t/large-protein-western-blot/873/1 "2025-02-11T11:04:47Z")

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Hi everyone

I have a problem with Western Blot of Large Protein (Transmembrane receptor 500kDa). I am using 3-8% Tris-Bis Gel and DTT in my loading buffer. I found the protein could not run down the Gel properly. I was advised that not boiling the sample will help. Is that true?

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**Author:** ![Navneet](https://avatars.discourse-cdn.com/v4/letter/n/8e8cbc/32.png) [@Navneet](https://scientistsolutions.discourse.group/u/Navneet)\
**Post date:** [February 11, 2025, 6:54pm UTC](https://scientistsolutions.discourse.group/t/large-protein-western-blot/873/2 "2025-02-11T18:54:46Z")

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Sarah This is true because:

- Large transmembrane proteins, especially those rich in **hydrophobic regions** , tend to **aggregate and precipitate** when boiled which prevents them from properly entering and running down the gel.
- Large proteins require **gentle denaturation** to maintain solubility.
- Boiling may cause them to **misfold or aggregate** rather than fully denature.

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**Author:** ![GAGAN](https://yyz2.discourse-cdn.com/flex004/user_avatar/scientistsolutions.discourse.group/gagan/32/16_2.png) [@GAGAN](https://scientistsolutions.discourse.group/u/GAGAN)\
**Post date:** [February 11, 2025, 7:00pm UTC](https://scientistsolutions.discourse.group/t/large-protein-western-blot/873/3 "2025-02-11T19:00:51Z")

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Sarah

Try the following:

1. Heat at 50–60°C for 5–10 minutes instead of boiling (95°C). This **partially denatures** the protein while keeping it soluble.
2. **Use Urea or SDS for Denaturation:** You can entirely omit the boiling step and replace it with addition of **6–8M urea** or increase **SDS concentration** (0.2–0.5%) in the loading buffer.
3. Try a **Stronger Reducing Agent** Other Than DTT e.g. **TBP or TCEP**
4. Use a **Lower Voltage for Running the Gel** as this will allow the larger molecules to stack more effectively and help these enter the gel.